
(W^ Baa 

Book. M e 4 



Copyright ft . 



Q 



_ 



CDFiRIGHT DEPOSIT. 



PATHOLOGICAL 
TECHNIQUE 

A Practical Manual for Workers in 
Pathological Histology and Bacteriology 

— including ■ 

Directions for the Performance of Autopsies and 
for Clinical Diagnosis by Laboratory Methods 

BY 

FRANK BURR MALLORY, A-M-, M.D. 

Associate Professor of Pathology, Harvard Medical School; Pathologist 
to the Boston City Hospital 

AND 

JAMES HOMER WRIGHT, A.M„ M,D V S-D. 

Pathologist to the Massachusetts General Hospital; Assistant Professor of 
Pathology, Harvard Medical School 



Seventh Edition 
Revised and Enlarged 
With 181 Illustrations 



PHILADELPHIA AND LONDON 

W- B- SAUNDERS COMPANY 
I9ta 






^ 






Copyright, 1897, by W. B. Saunders. Reprinted September, 1898. Revised, 
printed, and recopyrighted July, 1901. Revised, reprinted, and recopyngnted 
July, 1904. Revised, reprinted, and recopyrighted July, 1908. Re- 
vised, reprinted, and recopyrighted July, 191 1. Reprinted July, 
1913. Revised, reprinted, and recopyrighted April, 1915. 
Reprinted March, 1918. Revised, reprinted, and re- 
copyrighted October, 1918 



Copyright, 1918, by W. B. Saunders Company 



©cusosnaa 

NOV -2 1313 



PRINTED IN AMERICA 



PRESS OF 

fi. B. SAUNDERS COMPANY 

PHILAPEUPHIA 



It*./, 



- 



TO 

HENRY F. SEARS, A. M., M. D., 

WHO BY HIS LIBERALITY FIRST RENDERED POSSIBLE PATHOLOG- 
ICAL RESEARCH IN BOSTON, AND BY HIS PERSONAL 
WORK ADVANCED AND STIMULATED IT, 

THIS BOOK IS RESPECTFULLY DEDICATED BY 

THE AUTHORS 



PREFACE TO THE SEVENTH EDITION 



The book has been rearranged in this edition with the 
object of making it more useful. Among other changes 
the postmortem technique has been put at the end. The 
revision has been fairly thorough, but not so complete as 
could be wished owing to the war. 

Of additions, the following deserve mention: Goodpas- 
ture's acid polychrome methylene-blue stain for frozen 
sections of fixed tissues and also for demonstrating meta- 
chromatically the different granules in the islet and acinar 
cells of the pancreas; Graham's oxidase stain for the gran- 
ules in the myeloblastic series of cells and leukocytes; 
Benians' Congo red method for the demonstration of 
spirochetes; Claudius' stain for flagella; and the approved 
method of classifying pneumococci with reference to 
serum treatment. 

As useful procedures which hitherto have not been gen- 
erally recognized, attention is called to the use of the 
safety-razor blade in section cutting, the employment of 
benzene in paraffin embedding, and Rubaschkin's method 
of fixing celloidin and frozen sections to the slide for 
staining. 

Boston, Mass., October, 191 8. 

9 



PREFACE TO THE FIRST EDITION. 



This book is designed especially for practical use in 
pathological laboratories, both as a guide to beginners and 
as a source of reference for the advanced. We believe that 
the book will also meet the wants of practitioners who have 
more or less opportunity to do general pathological work. 

Every autopsy presents for solution a problem which may 
be simple or complex. The known quantities are certain 
clinical symptoms and physical signs ; the unknown quan- 
tities are not only the gross and microscopic lesions that 
may or may not have given rise to the symptoms and signs, 
but also the etiology of the lesions and the order of their 
sequence. The solution of the problem often requires the 
highest skill in post-mortem, bacteriological, and histo- 
logical technique, but in its solution lies the fascination of 
pathological work. 

It has seemed advisable to us to present, so far as possible, 
a consecutive statement of the methods employed in solving 
the various problems that arise, so as to avoid the repetitions 
that necessarily occur when the three usual divisions of the 
subject are separately considered by different writers. It 
is hoped that this method of presenting the subject will 
bring the student to the realization that the mechanical per- 
formance of a post-mortem examination and the inspection 
of the gross lesions constitute usually only the beginning 

of the solution of the problem, which should be investigated 

11 



12 PREFACE. 

bacteriologically, histologically, and chemically as far as our 
present knowledge will permit. 

We should particularly advise the routine bacteriological 
and histological examination of the more important organs 
in all suitable cases. Naturally, the autopsies in which the 
lesions are due to a single etiological factor are the most 
valuable and instructive for a clear understanding of the 
pathological processes present. 

Besides the methods of post-mortem examinations and 
of bacteriological and histological investigations connected 
with autopsies, we have added the special methods em- 
ployed in clinical bacteriology and pathology. 

In the parts devoted to Bacteriology and to Pathological 
Histology we have not endeavored to make an exhaustive 
collection of methods and formulae, but rather to select 
those which have been found of the greatest service in 
practical work. 



CONTENTS 



HISTOLOGICAL METHODS. 

Introduction, 17 — Laboratory Outfit: Microscopes, 18; Microtomes, 20; 
Centrifuge, 25; Vulcanized Fiber, 26; Knives, 27; Running Water, 28; Slides, 
28; Cover-slips, 29; Staining Dishes, 29; Metal Instruments, 31; Bottles, 31; 
Incubator, 31; Storage of Microscope Slides, 31; Examination of Fresh 
Material, S3 — Indifferent Fluids, 35 — Macerating Fluids, 35 — Examination 
of Fluids, 35 — Injections, 36 — Cold Injection-masses, 36 — Warm Injection- 
masses, 37— Fixing Reagents, 38 — Alcohol, 39 — Formaldehyde, 41— Alcohol 
and Formaldehyde, 43 — Corrosive Sublimate, 43 — Giemsa's Corrosive Sub- 
limate-alcohol Fixative, 44 — Zenker's Fluid, 45 — Chrome Salts, 46 — 
Miiller's Fluid, 46 — Orth's Fluid, 47 — Osmic Acid, 48 — Flemming's Solution, 
48 — Marchi's Fluid, 48 — Hermann's Solution, 48 — Pianese's Solution, 49 — 
Boiling, 49 — Decalcification, 49 — Directions for Using Nitric Acid, 50 — 
Phloroglucin and Nitric Acid, 51 — Sulphurous Acid, 52 — Trichloracetic Acid, 
52 — Frozen Sections, 52 — Wright's Embedding Method for Frozen Sections, 
54 — Celloidin and Paraffin Embedding Processes, 55 — Celloidin, 56 — 
Embedding in Celloidin, 56 — Paraffin, 58 — Embedding in Paraffin, 59 — 
Mayer's Glycerin-albumin Mixture, 62 — Serial Sections, 63 — By the Cel- 
loidin Method, 63 — By the Paraffin Method, 66 — Staining Solutions: Hema- 
toxylin and Hematein Stains, 66 — Carmine Stains, 71 — Aniline Dyes, 73 — 
Diffuse Stains, 78 — Combination Stains, 79 — Pianese's Staining Solutions 
and Staining Methods, 80 — Orcein, 83 — Iodin, 83 — Lugol's Solution, 83 — 
Staining Methods, 84 — Nuclear Stains, 87 — Aqueous Alum-hematoxylin, 88 
— Mallory's Instantaneous Alum-hematoxylin, 88 — Delafield's Alum-hema- 
toxylin, 88 — Harris's Alum-hematoxylin, 88 — Mayer's Hemalum, 89 — 
Heidenhain's Hematoxylin Stain, 89 — Weigert's Iron Hematoxylin, 89 — 
Heidenhain's Iron Hematoxylin, 90 — Mallory's Chlorid of Iron Hematoxylin, 
90 — Carmine Stains, 92 — Aniline Dyes as Nuclear Stains, 93 — Diffuse or 
Contrast Stains, 95— Combination Stains, 96 — Staining in Mass, 97 — 
Mitosis, 97 — Directions for Staining Karyomitotic Figures with Safranin, 99 
— Metallic Stains or Impregnations, 99 — Silver, 99 — Gold, 101 — Osmic Acid, 
102 — Clearing Reagents, 103 — Mounting Reagents, 105 — Special Stains for 
Certain Tissue-elements Other than Nuclei, 107 — Mitochondria, 107 — 
Mast-cells, 108 — Plasma-cells, 109 — Schridde's Method for Demonstrating 
Granules (Mitochondria?) in the Cytoplasm of Plasma-cells and Lympho- 
cytes, no — The Collagen Fibrils and Reticulum of Connective Tissue, in — 
Fibroglia Fibrils, 114— Elastic Fibers, 115 — Smooth and Striated Muscle- 
cells, 119 — The Central Nervous System, 120 — General Stains, 122 — Stains 
for Nissl or Tigroid Bodies, 124 — Ganglion-cells; Dendritic and Axis-cylinder 
Processes, 125 — Axis-cylinders and their Terminal Processes, 129 — Stains 
for the Myelin-sheath, 135 — Stains for Neuroglia-fibers, 142 — Degenerations 
of the Nervous System, 146— Methods of Fixing and Examining Special 
Organs and Tissues, 147 — Acute Inflammatory Exudations; Granulation- 
tissue, 148 — Lung, 149 — Bone-marrow and Spleen, 149 — Kidney, 153 — 
Gastro-intestinal Tract, 154 — Liver, 154 — Pancreas, 155 — Bone and Carti- 
lage, 157 — Skin, 160 — Museum Preparations, 162 — Pathological Products, 
164 — Cloudy Swelling; Albuminous Degeneration, 164 — Fat, 165 — Necrosis, 
169 — Caseation, 170 — Fibrin, 170 — Mucin, 171 — Psuedo-mucin, 173 — 
Colloid and Hyalin, 174 — Glycogen, 177 — Amyloid Infiltration, 180 — Pig- 
mentation, 183 — Petrifaction, 187. 

13 



14 CONTENTS. 



CULTURE=MEDIA. 

The Preparation of Test-tubes, 189 — Preparation of Culture-media: 
Bouillon, 190 — Glucose or Dextrose Bouillon, 192 — Agar-agar (Plain), 193 — 
Glucose Agar-agar, 196 — Glycerin Agar-agar, 196 — Blood agar, 197 — Glu- 
cose or Dextrose Gelatin, 198 — Blood-serum ((Loffler's Mixture), 198 — 
Litmus-milk, 201 — Potato-cultures According to Bolton, 201 — Dunham's 
Pepton Solution, 202 — The Adjustment of the Reaction of Culture-media by 
Titration, 202 — The Filling of Test-tubes, 204 — Sterilization of Culture- 
media, 206 — The Autoclave, 207 — The Storage of Culture-media, 208. 



CULTURE METHODS. 

Methods of Collecting Material, 209 — Examination by Cultures, 212 — 
Method of Preparing Cultures on Blood-serum, 2 1 2 — The Platinum Wire or 
Loop, 213 — Methods of Obtaining Pure Cultures, 216 — Method of Isolation 
of a Bacterium in Pure Culture from a Mixed Growth, 217 — Cultivation 
without Oxygen (Anaerobic Cultures), 220 — Culture-media for Anaerobic 
Bacteria, 221; Method of Liborius, 221 — Simple Anaerobic Plate-cultures, 
223 — Buchner's Method, 223 — Hans Zinsser's Method for Anaerobic Plate- 
cultures, 224 — Wright's method, 225 — The Determination of the Motility 
of Bacteria, 227; Preparing a Hanging Drop, 227 — Hill's Hanging-block 
Method for the Observation of Developing Bacteria, 228 — The Inoculation 
of Animals, 229 — Guinea-pigs, 229 — Rabbits, 230 — Mice, 231 — The Care of 
Animals, 233. 



METHODS OF STAINING BACTERIA. 

The Staining of Bacteria in Smear Preparations, 234 — Simple Staining, 235 
— Pappenheim's Pyronin and Methyl-green Mixture, 236 — Gram's Method 
of Staining, 236 — W. H. Smith's Method of Staining Bacteria in Sputum, 237 
— The Staining of Spores, 238; Abbott's Method, 238 — Moeller's Method, 
238 — Fiocca's Method, 239 — The Staining of Flagella, 239; Loffler's Method, 
240 — Williams' Method, 241 — Claudius' Method for Staining Flagella, 243 — 
Zettnow'-s Flagella Staining Method, 244 — The Staining of Capsules of Bac- 
teria, 244; W. H. Smith's Method for Smears, 244 — Hiss's Method, 245 — 
Richard Muir's Method, 246 — The Staining of Bacteria in Sections, 246— 
Pathogenic Bacteria which do not Stain by Gram, 248; Loffler's Methylene- 
blue Stain for Bacteria, 248 — Methyl-green-pyronin Stain, 249 — Pathogenic 
Bacteria which Stain by Gram, 249; The Gram Staining Method, 249 — The 
Gram-Weigert Staining Method, 250 — VerhoefT's Modified Gram Stain for 
the Leptothrix of Parinaud's Conjunctivitis, 250 — The Staining of Capsules 
in Sections, 251 — W. H. Smith's Method for Sections, 251. 



PATHOGENIC BACTERIA AND FUNGI. 

Staphylococcus Pyogenes Aureus, 253 — Staphylococcus Pyogenes Albus 
and Staphylococcus Pyogenes Citreus, 256 — Staphylococcus Epidermidis 
Albus, 256 — Staphylococcus Cereus Albus, 257 — Staphylococcus Cereus 
Flavus, 257 — Streptococcus Pyogenes and Allied Streptococci, 257 — Pneu- 
mococcus, 263 — The Determination of Types of Pneumococcus, 268 — Strep- 
tococcus Capsulatus, 270 — Gonococcus, 272; Special Culture-media, 273 — 
Diagnosis, 274 — Method of Staining for Gonococci, 276 — Micrococcus 
Catarrhalis, 277— Micrococcus Tetragenus, 278 — Diplococcus Intracellularis 
Meningitidis, 279 — Bacillus of Influenza, 282 — Bacillus Pertussis (Bordet- 
Gengou), 286— Bacillus Coli Communis, 288— Bacillus of Typhoid Fever, 
293; The Blood-serum Reaction in Typhoid Fever; Widal Reaction, 297 — 



CONTENTS. 1 5 

Cultivation of the Typhoid Bacillus from the Blood during Life, 298 — Para- 
typhoid Bacilli, 299; Differential Diagnosis between the Bacillus of Typhoid 
Fever and the Bacillus Coli Communis, 299 — Bacillus Dysenteriae, 300 — 
Examination of Feces for Typhoid, Paratyphoid, and Dysentery Bacilli, 
302; Preparation of Media, 302 — Bacillus Proteus (Proteus Vulgaris), 304 — 
Bacillus Diphtherial, 304; Special Methods of Staining the Bacillus Diph- 
therias, 309 — Mallory's Stain for the Diphtheria Bacillus, 311 — Scarlet Fever, 
312 — Bacillus of Glanders (Bacillus Mallei), 314; To Stain the Glanders 
Bacillus in Sections, 318 — Lonier's Methylene-blue Stain for Sections, 318 — 
Schutz's Method, 318 — Noniewicz's Method, 319 — Bacillus of Chancroid 
(Bacillus of Ducrey), 319; Method of Isolation from the Lesions, 321 — 
Bacillus Pyocyaneus (Bacillus of Green Pus), 321— Bacillus of Bubonic 
Plague, 323 — Bacillus of Anthrax, 327; Diagnosis, 330 — Bacillus Mucosus 
Capsulatus, 331 — Bacillus of EJiinoscleroma, 334 — Bacillus Aerogenes Cap- 
sulars, 334 — Bacillus of Tetanus, 336 — Bacillus Aerogenes Capsulatus, 340 
— Bacillus Tuberculosis, 342; Diagnosis, 347 — Examination of Sputum for 
Tubercle Bacilli, 348 — Tubercle Bacilli in Urine, 350; Tubercle Bacilli in 
Tissues, Pus, and Feces, 350 — Bacteria that Stain by the Tubercle Bacillus 
Method, 351 — Ehrlich's Method, 352 — Ziehl-Neelson-Gabbet Method, 352 
— Kuhne's Method, 352 — To Stain Tubercle Bacilli in Celloidin Sections, 
353 — Bacillus of Leprosy, 353 — Spirillum of Asiatic Cholera (Comma Bacil- 
lus), 355; Bacteriological Diagnosis, 361— The Micro-organism of Actino- 
mycosis, 362; Cultures, 367 — Pathogenesis, 368 — Method of Isolation, 368 — 
To Stain the Actinomyces in Sections, 371 — Mallory's Stains, 372 — Sporo- 
trichum Schenckii, 373 — The Blastomycetes, 376 



ANIMAL PARASITES. 

Entamoeba?, 381; The Cultivation of Entamoeba:, 384 — Malarial Organ- 
isms, 385 — Giemsa's Method for Staining Protozoa and Bacteria in Sections, 
392 — Rabies (Hydrophobia), 394 — Spirochetes of Relapsing Fever, 396 — 
Syphilis, 397 — Tape-worms, 403 — Taenia Solium, 404 — Taenia Mediocanellata 
s. Saginata, 405 — Taenia Echinococcus, 405 — Dibothriocephalus Latus, 405 
— Schistosoma Haematobium (Distomum Haematobium, Bilharzia), 405 — 
Round-worms, 406 — Trichinellae, 408. 



CLINICAL PATHOLOGY. 

Examination of the Blood, 410 — Method of Counting the Red and White 
Blood-corpuscles, 410 — Wright's Method of Counting the Blood-platelets, 
414— Cover-glass Preparations, 415 — Methods of Staining, 417 — Method 
of Examining Blood without Drying or Fixation, 421 — Schultze's Oxydase 
Reaction, 422 — Graham's Alphanaphthol-pyronin Stain for the Oxydase 
Granules, 423 — Cultures from the Blood during Life, 425 — Mcjunkin's Tube 
for Collecting Blood for Cultures, 425 — Serum Diagnosis of Syphilis by 
Means of the Wassermann and Noguchi Reactions, 427 — The Complement- 
fixation Test in Gonorrheal Infections, 445 — The Complement-fixation Test 
in Echinococcus Infection, 446 — Examination of the Cerebrospinal Fluid, 
447 — Protein Increase Tests, 449 — Alzheimer's Method for the Cytological 
Examination of the Cerebrospinal Fluid, 449 — Lange's Colloidal Gold Test 
of the Cerebrospinal Fluid for Syphilis of the Central Nervous System, 451 — 
Examination of Tissues and Fluids, 454 — Uterine Scrapings, 454 — Examina- 
tion of Fluids Obtained by Puncture, 455 — Examination of Serous Fluids, 
456 — Cytodiagnosis, 457 — Animal Inoculation, 461 — Ovarian and Parova- 
rian Cysts, 461 — Pancreatic Cyst or Fistula, 462 — Dropsy of the Gall-bladder, 
462 — Hydronephrosis and Renal Cysts, 462 — Echinococcus Cysts, 463 — 
Examination of the Sputum, 463 — Examination of the Gastric Contents, 469 
— Examination of the Feces, 470 — Examination of the Urine, 471. 



1 6 CONTENTS. 



POST=MORTEM EXAMINATIONS. 

Introduction, 472 — Instruments, 473 — General Rules, 477 — Suggestions to 
Beginners, 478 — Private Autopsies, 479— External Examination of the Body, 
481 — Inspection of the Body as a Whole, 481— Special Inspection of the 
Different Parts of the Body, 482 — Internal Examination of the Body, 482 — 
Opening of the Abdominal Cavity, 483 — Inspection of the Abdominal Cav- 
ity, 484 — Opening of the Thorax, 485 — Inspection of the Pleural Cavities, 
486 — Opening of the Pericardium, 487 — External Inspection of the Heart, 
487 — Opening of the Heart, 488 — Removal of the Lungs, 492 — Organs of the 
Neck, 494 — The Abdominal Cavity, 495 — The Spleen, 496 — The Gastro- 
intestinal Tract, 496 — The Liver, 499 — The Kidneys and Adrenals, 500 — 
The Pelvic Organs, 502 — Removal of the Brain, 505 — External Examination 
of the Brain, 510 — Section of the Brain, 511 — Removal of the Spinal Cord, 
515 — The Eye, 517 — The Ear, 518 — The Naso-pharynx, 519 — Examination 
of New-born and very Young Children, 520 — Restitution of the Body, 522. 

ADDENDA. 

Method of Preparing the Bacterial Vaccines of Sir A. E. Wright, 525— 
Methyl-violet Shellac, 529 — Method for Blackening Table Tops, 529 — To 
Clean Slides, 530 — Hand Lotion, 530 — Index, 531. 



Pathological Technique 



HISTOLOGICAL METHODS. 



Introduction. — The ideal function of the technique of 
pathological histology is so to fix tissues for microscopic 
examination that every tissue-element or pathological prod- 
uct is perfectly preserved with all its morphological and 
chemical properties intact, and so to stain tissues that every 
tissue-element or pathological product can be readily differ- 
entiated from any other tissue-element or pathological prod- 
uct that resembles it. In certain respects only has this ideal 
yet been reached, but the number of differential stains is 
increasing yearly. 

In the following pages the various steps in the prepara- 
tion and staining of tissues have been arranged, so far as 
possible, in logical sequence. 

LABORATORY OUTFIT. 

The modern pathological laboratory, especially if con- 
nected with a hospital, requires in its outfit a considerable 
number of instruments and utensils owing to the variety 
of work which must be performed in it. It is not our func- 
tion to appraise the relative merits of the different micro- 
scopes and microtomes, for example, which are on the 
market, but we shall mention certain ones which we have 
found from long-continued use to be most excellent. Amer- 
ican microscopes are steadily improving, but have not yet 
reached the standard set by Zeiss, although more nearly 
approaching it every year. In other lines, such as micro- 



1 8 PATHOLOGICAL TECHNIQUE. 

tomes, incubators, and centrifuges, it is doubtful if our 
instruments can be excelled elsewhere. 

Microscopes. — The most important laboratory instru- 
ment is the microscope. It should be, so far as means will 
permit, the best that skill can produce. Excellent micro- 
scopes are manufactured both abroad and in this country, 
but no make of microscope can be unconditionally recom- 
mended. Undoubtedly the best microscopes in every par- 
ticular and the most expensive are those manufactured by 
Zeiss. 

At the present time, however, they are unobtainable. 
We shall have to be satisfied with what we can get in our 
home market. Microscopes made in the United States are 
better than they used to be and are steadily improving, but 
they are not yet perfect. Those manufactured by the 
Bausch & Lomb Optical Co. can be highly recommended. 

It is important for a beginner in microscopy, before buy- 
ing a microscope of any make, to have it carefully examined 
and its lenses tested at a pathological or other laboratory by 
some one skilled in its use. The continental form of stand 
of medium size is to be preferred to all others. The large 
stand is undesirable, because it is too heavy and too high for 
comfortable use. It should be furnished with rack and pin- 
ion, and with micrometer screw for coarse and fine adjust- 
ment, with a triple or quadruple nose-piece, and with an 
Abbe condenser and iris diaphragm. The necessary ob- 
jectives are a low and a high dry, and a T V oil-immersion. 
Two eye-pieces, a low and a high, will be found sufficient 
for all ordinary purposes. 

The stands, oculars, and objectives of the Zeiss make 
generally used are the following: 

Stands, III and IV. 

Oculars, 2 and 4. 

Objectives, AA, D, and ^ oil-immersion. 

Or in the apochromatic series, 

Oculars, 4, 6, and 8. 

Objectives, 16.0, 8.0, 4.0, and oil-immersion 2.0 mm., 
apert. 1.30. 



HISTOLOGICAL METHODS. 1 9 

Even if all these different parts cannot be purchased at 
the same time, it is important to buy a stand to which they 
afterward may be added, for the list includes only what every 
medical practitioner should have at his service for the proper 
examination of urine, sputum, blood, etc. 

The apochromatic lenses and compensation oculars are 
too expensive to come into general use. Fortunately, they 
are more important for photomicrography than for general 
microscopic work. 

The oil-immersion le?ts should always be cleaned after 
using by wiping off the oil with an old linen or silk hand- 
kerchief or with the fine lens-paper now manufactured for 
that purpose. If the lens is sticky, moisten the cloth with 
benzol or xylol. The same process can be used if necessary 
for the dry lenses, but it must be done quickly, so as not to 
soften the balsam in which the lenses are imbedded. Ordi- 
narily a dry cloth is sufficient. 

In using the Abbe illuminating apparatus it is important 
to bear in mind that the best results are obtained, according 
to Zeiss, by employing the plain mirror, for the condenser is 
designed for parallel rays of light. The concave mirror is 
to be used only when some near object, such as the window- 
frame, is reflected into the field of vision or when artificial 
light is employed. 

A mechanical stage is now made which can be instantly 
attached to any microscope. It is exceedingly useful for 
blood-counting or for searching carefully the whole surface 
of a stained cover-slip. For ordinary work it is undesirable. 

Illumination.— For microscopic work the best illumina- 
tion is that obtained from a white cloud, although for some 
purposes the light which filters through a white curtain on 
which the sun is shining is very useful, especially with the 
highest powers of the microscope. When artificial light is 
necessary, the Welsbach burner, or, better still, a Tungsten 
electric light with ground-glass globe, will be found very 
satisfactory. The slightly yellowish tint of the light can be 
corrected, if necessary, by means of a piece of blue glass or, 
better still, of the new daylight glass inserted beneath the 



20 PATHOLOGICAL TECHNIQUE. 

Abbe condensor. Of the different electric lamps designed 
for use with the microscope that put out by Leitz is so far 
the simplest, best, and most powerful. It is advisable to 
use the large form designed for dark-field illumination and 
furnished with the ioo Watts nitrogen filled tungsten bulb 
so as to be able to obtain plenty of light for oil-immersion 
work. Inasmuch as the large daylight glass filter which 
goes with it is very liable to break, it seems better to re- 
place it with the ground glass and to insert a small circle 
of daylight glass below the Abbe condensor. 

For drawing, the Abbe camera lucida will be found ex- 
tremely useful and convenient. 

Much use is also made of a vertical projection apparatus 
for the same purpose, especially when only outline drawings 
are required. For fine details it is not so good. 

Microtomes. —Three different kinds of microtomes are 
required in laboratory work. They are known as the freez- 
ing microtome, the celloidin microtome, and the paraffin 
microtome. Each has its own special field of usefulness. 

Freezing Microtome. — Freezing by means of the evapora- 
tion of ether, more rarely of rhigolene, was originally the 
method in general use. The process was both expensive 
and slow. A much cheaper and more rapid method of 
freezing was originated many years ago in the Pathological 
Laboratory of the Harvard Medical School by Dr. S. J. 
Mixter, and has since been universally adopted. This 
method consists in the employment of compressed carbon- 
dioxid, which is found in commerce in iron cylinders con- 
taining each about twenty pounds of liquefied gas. It is 
commonly used for charging beer and soda-water. As a 
rule, the cylinders are loaned, so that it is necessary to pay 
for the contents only. 

The cylinder must be securely fastened in an upright 
position near the microtome, with its valve end below and 
with its escape-tube on a level with the entrance-tube into 
the freezing-box. The cap covering the escape-tube of the 
cylinder should have a small hole bored through it, and into 
this hole a small brass tube about 5 cm. long, with a fine 
bore, should be tightly driven. This permits the use of a 



HISTOLOGICAL METHODS. 21 

smaller stream of gas than the escape-pipe of the cylinder 
would otherwise furnish. The same cap can be kept to use 
on all future cylinders. 

The cylinder is connected with the microtome by means 
of a short piece of thick, strong rubber-tubing with small 
bore, so as to fit snugly over the escape-tube of the cylinder 




Fig. i. — Freezing microtome. 

and the entrance-tube into the freezing-box. It is advisable 
to wire each end of the rubber-tubing around the tube it 
incloses. The connection can also be made with flexible 
copper tubing. 

In order to obtain better leverage and more perfect con- 
trol over the escape of the gas than are needed for the 
purposes for which the cylinders are ordinarily used, it is 
necessary to lengthen to about 25 cm., in whatever way 



22 PATHOLOGICAL TECHNIQUE. 

seems best, the handle of the key which opens the escape- 
valve. 

The first time the cylinder is used for freezing, a little 
water may escape, causing considerable sputtering. In 
freezing, the valve should be turned carefully, so that the gas 
may escape slowly and evenly. Tissues fixed by alcohol or 
any other reagent, except formaldehyde, must be washed in 
running water for some hours before they can be frozen. 

Even for tissue fixed in formaldehyd washing in water for 
ten to thirty minutes is advisable, as better sections can be 
obtained. 

It is now possible to obtain from stores carrying automo- 
bile supplies small tubes of compressed carbon dioxid suf- 
ficient for one or two freezings. They will be found con- 
venient for carrying to private operations when an immediate 
diagnosis by means of frozen sections is demanded. 

Several forms of the freezing microtome are on the market. 
Of the simple types, the Bausch & Lomb table microtome, 
No. 3050, with freezing attachment No. 3082, can be recom- 
mended. 

For cutting frozen sections on this type of instrument the 
blade of a carpenter's plane, 2§ inches wide, mounted in a 
wooden handle (Fig. 2), will be found very serviceable and 
easy to sharpen. 




Fig. 2. — Knife for freezing microtome, made from the blade of a carpenter's plane. 

Of the more complicated freezing microtomes that manu- 
factured by the Spencer Lens Co. can be highly recom- 
mended. 

Celloidin Microtome. — There are two types of celloidin 
microtomes — one in which the object is raised by a screw, 
a second in which the object is raised by being moved up an 
inclined plane. The first type of machine is the better, for 
two reasons: the screw affords greater accuracy in the even 
elevation of the object than is possible with an inclined 



HIS TO L O GICAL ME THODS. 



23 



plane, and the object remains at all times in the same rela- 
tive position with regard to the knife, so that an equally long 
sweep of the blade can be obtained for every section. An 




Fig. 3. — Large laboratory microtome (Bausch & Lomb). 

excellent instrument of this type is made by Bausch & 
Lomb (Fig. 3) . For practical work it is much to be preferred 




Fig. 4. — Minot precision microtome (Bausch & Lomb). 

to the elaborate Schiefferdecker- Becker microtome, designed 
for cutting sections under alcohol. 

A new and wholly original microtome, in which the knife 



24 PATHOLOGICAL TECHNIQUE. 

remains fixed and is clamped at both ends, while the object- 
holder, which is raised by a screw, moves back and forth 
beneath the knife, has recently been designed by Dr. C. S. 
Minot and is manufactured by Bausch & Lomb (Fig 4). 
It is intended both for celloidin and for paraffin work. 

A drop-bottle on an elevated stand, with screw arrange- 
ment for regulating the amount of alcohol, is the most con- 
venient method for keeping the object and the knife wet 
while cutting; 80 per cent, alcohol should be used. 

Paraffin Microtome. — Although paraffin sections can be 
cut on a celloidin microtome, it is preferable to have an 
instrument designed for the purpose. Two models of the 




Fig. 5. — Minot's wheel microtome (Bausch & Lomb). 

Minot wheel microtome are manufactured in this country: 
one by the Bausch & Lomb Optical Co., the other by the 
International Equipment Co., of Cambridge, Mass. The 
latter instrument has this advantage for pathological work: 
it is simple and heavy in construction, and the paraffin block- 
holder is controlled by a ball-and-socket joint, requiring but 
one screw instead of three. It has been found very satisfac- 
tory in practical use. 

Paraffin Bath. — The best bath for keeping paraffin at a 
constant temperature is a thermostat of suitable size with 
hot- water jacket, such as is used for growing cultures of 



HISTOLOGICAL METHODS. 



25 



bacteria. The paraffin is kept in it on shelves in glass dishes 
of various sizes. The advantages of this method over the 
old way of using copper cups set into the top of a water- 
bath are that the paraffin is kept absolutely free from dust, 
each worker can have his own set of dishes, and the smallest 
bits of tissue can be readily found in them, because they are 
transparent. 

A preliminary bath of soft paraffin is wholly unnecessary, 
and only prolongs the objectionable stage of heating. The 
regulator should register only one or two degrees above the 
melting-point of the paraffin. 




Fig. 6. — Minot rotary microtome (International Equipment Co., Cambridge, 

Mass.) . 



Paraffin should be melted and decanted or filtered before 
use, as it often contains foreign material. When hot, it runs 
through an ordinary filter without trouble. A hot-water 
jacket to the funnel is not at all necessary. 

Centrifuge. — This instrument is of great use in obtaining 
quickly the sediment from various fluids, including blood and 
urine, and also for sedimenting and washing the red blood- 
corpuscles used in the Wassermann and Noguchi serum 
tests. The electrically run instruments manufactured by the 
International Equipment Co., of Cambridge, Mass., can be 
highly recommended as well made, durable, and easy run- 



26 



PA THOL O GICAL TE CHNIQ UE. 



ning. Size I, type B, with 8-tube, 50 c.c. head will be found 
very satisfactory. 




Fig. 7. — Centrifuge, size i, type B, with 8-tube, 50 c.c. head manufactured by 
the International Equipment Co. 

Vulcanised Fiber. — For mounting celloidin prepara- 
tions nothing is so poor as cork, although it has been in use 
for years. The chief objections to it are that it does not fur- 
nish a rigid support to the imbedded object; that, unless 
weighted, it floats in alcohol with the specimen downward ; 
and that it yields a coloring material which stains both the 




Blocks of vulcanized fiber. 



alcohol and the specimen. Wood is not much better, 
although, of course, much firmer. Glass blocks have been 
proposed, and might do fairly well if there did not exist an 



HISTOLOGICAL METHODS. 



27 



ideal substance- 



in boards or strips, preferably J or -$ 



viz. , vulcanized fiber. This can be obtained 
& inch in thickness and 
sawn to any desired dimensions. It is perfectly rigid, is 
heavy enough to sink specimens to the bottom of the jar in 
an upright position, is unaffected by alcohol or water, is light 
red in color, so that it is easily written on with a lead pencil, 
gives off no coloring material, and is practically indestructible. 
Several parallel cuts, 1 to 2 mm. in depth, should be sawn 



<C-^- 




Fig. 9. — Diagram of the direction of the movements in honing. 

into the upper surface of each block, so as to give the celloidin 
a firm hold. 




fffWWff/MHtllllllllH WHiiHIIIIHIIHfHf llfmillfMfggflttWf f rmfifffftfilil I tlllllHI iih III I HI Ifff.V f if f/rt|- llll 



MPs 

) 

/ 

Fig. 10. — Diagram of the direction of the movements in stropping. 

Knives. — The knives for both the celloidin and the 
paraffin microtomes should be heavy and not too long, so as 
to afford as great rigidity as possible ; they should be bicon- 
cave, so that they may be easily sharpened. It is important 



28 PATHOLOGICAL TECHNIQUE. 

that every one who does much work in a pathological labor- 
atory should learn to sharpen his own knives. The requisite 
skill is not difficult to acquire, and the time spent in learning 
is fully compensated for by the ability always to have a sharp 
knife when it is wanted. For honing a knife either a fine 
water-stone or a glass plate with diamantine and Vienna 
chalk may be used. In honing, the edge of the knife is for- 
ward and the motion is from heel to toe. The knife should 
always be turned on its back, and the pressure on it should 
be at all times rather light. 

In stropping, the movement is reversed. The back of the 
knife necessarily precedes the edge, and the motion is from 
toe to heel. The direction of the movements in honing and 
stropping is best illustrated by the diagrams (Figs. 9, 10). 

The condition of the cutting edge can be examined by 
drawing the knife flatwise across the low power field of the 
microscope. When the knife is properly sharpened the edge 
is smooth and even, without nicks. 

A razor-strop paste greatly facilitates the smoothing of 
the knife edge in stropping. 

Gillette Safety Razor blades, used with a suitable holder, 
give satisfactory paraffin sections. Success in the use of 
these blades depends on careful adjustment of the blade in 
the holder so that the edge of the holder bears exactly on the 
beginning of the bevel of the blade. A holder designed by 
J. H. Wright may be obtained from the International 
Equipment Co. 

Running water for washing out specimens which have 
been fixed in Flemming and other solutions is most easily 
supplied by having a water-pipe, furnished with numerous 
cocks 5-10 cm. apart, run horizontally over a slightly slop- 
ing shelf adjoining the sink. Attached to each cock is a 
rubber tube, with a glass tube in the end of it long enough 
to reach to the bottom of the jar (Fig. 11). By this arrange- 
ment the amount ol water supplied to each specimen can be 
easily regulated. 

Slides should be of colorless glass with ground edges. 
The English form, measuring 1 X 3 inches (76 X 26 mm.), is 



HISTOLOGICAL METHODS. 29 

to be preferred for ordinary use. Occasionally broader slides 
are needed. Thick slides are preferable to thin ones; the 
latter are so light that they are easily lifted by the oil- 
immersion lens ; they also seem to warp when heated to 
attach paraffin sections to them. They break readily if too 
much pressure is applied in wiping or rubbing them. 



■ 

i 


f ■" # ==== ^ * J* 1 

Kiiiv'i ' ■ in' i"'iri ' -Ti 'it " r \^~ 


L - 


Pr*- ■ iff**!* 



Fig. 11. — Large laboratory sink, showing adjoining shelf and arrangement for 

running water. 

Cover-slipS should be square or oblong according to the 
shape of the specimen. Most dry lenses are adjusted for 
cover-glasses measuring 16 or 17/i in thickness, so that if 
possible no cover-slip ranging outside of 15 to 18/-* should 
be used. With an oil-immersion the exact thickness is not 
quite so important. 

Slides and cover-slips are cleaned by dipping in alcohol 
and wiping dry with a soft crash towel or old linen handker- 
chief. 

Coverslips, after they are clean, should be preserved dry 
in covered dishes. The common method of keeping them 
under alcohol cannot be recommended. 

Staining Dishes. — Watch-glasses are not satisfactory 
on account of their instability. Concave dishes with flat 



3° 



PA THOL O GICA L TE CHNIQ UE. 



bottoms are much better for ordinary use, and can be ob- 
tained of several patterns. They should be large enough 
to hold 25 c.c. of fluid. The Syracuse solid watch-glasses 
are very good dishes of this shape. Individual glass butter 
dishes can be obtained which are very satisfactory and com- 
paratively cheap. 

Slender dishes of various sizes will be found useful for 
many purposes. 

The "New Practical Staining Dish" (No. 16,618) manu- 
factured by the Bausch and Lomb Optical Co. is very useful 
for staining at once a number of paraffin sections. 

Oblong rectangular Petri dishes are very convenient for 
staining preparations mounted on the slide. 

For staining at once a large number of sections for class 
purposes, the Hobb's Tea Infuser has been found very useful, 
If set in a small lemonade cup, but little stain is required. 

Large concave dishes holding 200 c.c. will be found the 
most convenient for holding frozen sections of fresh tissue, 
because a slide can be dipped into them and under the 
sections. They are known in the trade as glass nappies. 





Fig. 12. — New practical staining dish. 

Large flat-bottomed glass dishes known as crystallizing 
dishes, holding one to three liters, are excellent to fix tissues 
in, as they allow the thin slices of material to lie flat. If 
several sizes are obtained the larger dishes serve as good 
covers for the smaller ones. 



HISTOLOGICAL METHODS. 3 1 

Metal Instruments. — Spatulas of different sizes are 




Fig. 13. — Spatula. 

needed. They should be thin, smooth, and large enough, 
so that a section will not curl over the edge (Fig. 13). 

The best instrument for transferring sections under all cir- 
cumstances is a piece of platinum wire mounted in an ordi- 
nary screw needle-holder. It is pliable and can be bent to 
any shape, will not break like a glass needle when dropped, 
and is not affected by acids. Ladies' hat-pins form a cheap 
but serviceable substitute. They are readily bent to any 
desired shape by heating. Forceps, scissors, scalpels, and 
many other instruments required in microscopical work do 
not need any special mention. 

Bottles. — For cover-slip work and for staining on the 
slide dropping-bottles will be found extremely convenient. 
The patent T. K. pattern of 50 c.c. capacity, with flat top, is 
probably the best form and size. 

For stains and reagents, glass- and cork-stoppered bottles 
of various sizes are required. The sizes most used are those 
containing 125, 250, 500, and 1000 c.c. 

The lightning jars of half-pint and pint capacity, such as 
are employed for preserving fruit, can be highly recommended 
for holding pathological tissues after fixation. Wide-mouthed 
IOO c.c. bottles are useful for holding small amounts of tissue. 

Incubator. — Incubators are needed for two purposes, 
for growing cultures of micro-organisms and for paraffin 
embedding. Owing to the high cost of metals the tendency 
at present is to replace the heavy waterjacketed copper 
structures of the past with simple ones made of wood and 
lined with asbestos. At the same time the system of keep- 
ing them at a constant temperature is steadily changing from 
gas to electricity because it has been found simpler and more 
reliable. 

Storage of Microscope Slides. — Microscope slides 



3 2 PATHOLOGICAL TECHNIQUE. 

may be stored in cabinets (the most expensive way), in 
boxes, or in trays. We have tried them all and use them all, 
but some of them have certain advantages to which we shall 
call attention. Wooden cabinets with shallow drawers in 
which the slides lie flat can be obtained of various capacities 
up to those which will hold nine thousand. The important 
point is to have the compartments in the drawers wide 
enough so that an individual slide can be turned around 
flatwise in them without binding at the edges (J inch deep, 
3 J inches wide, 15 inches long, more or less). On this 
account the Minot pressed tin cabinet cannot be recom- 
mended; the drawers are too shallow, the transverse ridges 
are a nuisance, and the lateral spacing is too narrow, so that 
a slide the least bit over length will not fit in it. Moreover, 
the drawers have a great tendency to stick in the case. 

In another type of cabinet the compartments in the 
drawers are 1 J inches deep, so that the slides, after the balsam 
is thoroughly hardened, may be stored compactly on edge 
in them like so many cards. The disadvantage of this 
method is that there is always the danger of balsam oozing 
out and sticking the slides together. 

Several styles of boxes holding one hundred slides each 
are on the market. They afford the cheapest, neatest, and 
most convenient method of permanent storage. The green 
covered wooden box made by Bausch & Lomb is light in 
weight, the grooves are wide enough to admit the thickest 
slides, the alignment is true, and there is a moderate amount 
of play allowed for slides over length. We have found them 
very satisfactory. 

Small wooden boxes holding twenty-five slides each are 
often convenient. The box marketed by Bausch & Lomb 
has the advantages of lightness, true alignment, and suffi- 
cient play to admit slides of various dimension so that they 
will not bind. Moreover, the box has a top which cannot 
be confused with the bottom part; there is no danger of lift- 
ing off the wrong half. 

Pressed paper trays of a capacity of twenty slides are very 
convenient for holding slides while the balsam is drying and 
before they are packed away. 



HISTOLOGICAL METHODS. 33 

EXAMINATION OF FRESH MATERIAL. 

Fresh tissues may be examined either in teased prepara- 
tions or in sections. 

Teased preparations are made by cutting out a very small 
bit of the tissue in question and dividing it as finely as pos- 
sible, by means of two sharp, clean needles, on a slide in a 
drop or two of some indifferent fluid, such as the normal 
salt solution. Teased preparations are often made, for in- 
stance, of the heart-muscle when fatty degeneration is sus- 
pected. If the tissue is soft, the cells are easily obtained 
by simply scraping the cut surface with the edge of the 
knife. 

Sections of fresh tissues can be made with a razor or with 
a double knife, but much the better way, at least for general 
diagnostic purposes, is to use frozen sections, which can be 
very quickly and perfectly made with the freezing microtome. 
The fresh sections are put into salt solution or into ordinary 
tap water in a glass dish large enough to permit of a slide 
being dipped into it, so that a section can be floated and 
spread out evenly on its surface. The slide is then carefully 
raised, the excess of fluid is wiped off, and a coverslip put 
on. 

If it is desired to stain the section, a few drops of Loffler's 
methylene-blue solution, diluted I to 3 with water, are poured 
over it after it is spread evenly on the slide. Stain for twenty 
seconds. Then float the section off in a dish of water and 
wash out the excess of stain. Remount the section on the 
slide, wipe away the excess of water, and drop a coverslip on 
the preparation. If sections of fresh tissues are put directly 
into a staining fluid in the ordinary manner, they pucker up 
and do not stain evenly. 

A 1 per cent, aqueous solution of thionin gives a very 
pretty differential staining of nuclei and connective-tissue 
fibrils. 

For demonstrating amyloid either Lugol's solution of 
iodin or methyl-violet followed by 1 per cent, acetic acid can 
be used. 
3 



34 PATHOLOGICAL TECHNIQUE. 

Sections of fresh tissue may be fixed, stained, cleared, and 
mounted in balsam by a slight modification of the method 
described for frozen sections. This modification con- 
sists in covering the section with 95 per cent, or absolute 
alcohol after it has been spread out evenly on the slide as 
described in the method referred to. The alcohol is to be 
dropped on the section carefully from a drop bottle, in order 
to avoid folding. After thirty seconds the alcohol is drained 
off and the section flattened out on the slide with blotting"- 
paper and further treated according to the method above 
mentioned. If the section is not treated with alcohol before 
blotting, it will adhere to the blotting-paper and not to the 
slide. 

Fresh preparations are often treated with chemicals for 
various purposes. Of these chemicals, acetic acid is the 
most generally useful in pathological work. It shrinks the 
nuclei and renders their outlines more distinct. It swells 
connective tissue, making it more transparent, so that the 
elastic fibers which are unaffected stand out distinctly. It 
precipitates mucin and dissolves or renders invisible the 
albuminous granules. Its main use as a reagent for fresh 
tissues is to demonstrate fat and to differentiate that sub- 
stance from albuminous granules. 

Acetic acid is ordinarily used in a 1 to 2 per cent, aqueous 
solution, a few drops of which are placed at one edge of the 
cover-slip, and then drawn beneath it by placing a piece of 
filter-paper on the opposite side. If in a hurry, however, 
stronger solutions, or even glacial acetic acid, may be used. 
Other reagents are of less importance, but are occasionally 
used. 

Osmic acid is sometimes employed in a 1 per cent, aque- 
ous solution to demonstrate fat, which it stains brown to 
black. 

An alcoholic solution of Scharlach R. is being used more 
and more for the same purpose. It stains fat orange to 
red. 

Hydrochloric acid in a 3 to 5 per cent, solution is used to 



HISTOLOGICAL METHODS. 35 

demonstrate calcification. Phosphate of lime is simply dis- 
solved, while from carbonate of lime bubbles of carbon- 
dioxid (C0 2 ) are set free. 

Indifferent Fluids. — Fresh tissues are usually examined 
in an isotonic salt solution — a 0.9 per cent, solution of com- 
mon salt in water. It has the advantage over water that 
tissues do not swell up so much in it, blood-corpuscles are 
unaffected, and the finer structures are better preserved. A 
very few drops of Lugol's solution added to the stock-bottle 
of salt solution will be found useful in preventing the growth 
of mould. 

Serous fluids, such as hydrocele fluid, are occasionally 
used. Artificial serum is made by adding 1 part of egg- 
albumin to 9 parts of normal salt solution. 

Macerating" fluids are little used in pathology. Occa- 
sionally, however, when tissues are tough, so that they cannot 
be readily teased apart, they are macerated in certain fluids 
which dissolve the substances that hold the different ele- 
ments together. The reagents most commonly used are the 
following : 

1. Ranvier's one-third alcohol is made by taking 1 part of 
96 per cent, alcohol and 2 parts of water ; twenty-four hours 
are usually enough. 

2. Very dilute solutions of chromic acid are recommended 

— ifa to sfo of I P er cent - 

3. jj per cent. Caustic PotasJi. — Tissues are macerated in 

a few minutes to one hour : they must be examined in the 
same fluid, because the cells are destroyed if the solution is 
weakened. 

Examination of Fluids. — Small fragments of tissue 
should be picked out with forceps. If much blood is ad- 
herent, wash the tissue well in salt solution. When the cell- 
ular elements are few in number, they are obtained with a 
pipette, just as in urine-work, after allowing them to settle 
at the bottom of the glass. A centrifugal machine will be 
found of great service when the sediment is slight. 



36 PATHOLOGICAL TECHNIQUE. 

INJECTIONS. 

Injections are not much used in pathology. The process 
is an art that requires much patience and considerable ex- 
perience. The purpose of an injection is to render vessels 
and vessel-walls more visible than under ordinary circum- 
stances. Transparent, deeply colored fluid mixtures are 
used, which will become hard in the vessels. Some injec- 
tion-masses are employed cold, others warm. The warm 
injection-masses contain gelatin, and are much more trouble- 
some to use, but give much the more perfect results. For 
coloring the mass carmine is the best material, because it is 
a permanent color. 

The instruments required are cannulas of various sizes and 
a syringe, or, better still, a constant-pressure apparatus. 

When a warm injection-mass is used, the bottle containing 
the mass must be placed in a water-bath and kept at a tem- 
perature of about 45 C. The organ or animal to be in- 
jected must likewise be placed in a water-bath of the same 
temperature. 

It is very important that in connecting the end of the tube 
carrying the injection-mass with the cannula inserted in the 
vessel no air-bubbles shall enter. When blood-vessels are 
to be injected, it is advisable to wash them out first with 
normal salt solution. 

Cold Injection-masses. — i. Blue Coloring Mass. 

Soluble Berlin blue, I ; 

Distilled water, 20. 

2. Carmine Injection-mass (Kollmann). — Dissolve I gram 
of carmine in 1 c.c. of strong ammonia plus a little water; 
dilute with 20 c.c. of glycerin. To this solution add 1 gram 
of common salt (NaCl) dissolved in 30 c.c. of glycerin. To 
the whole solution add an equal quantity of water. 

Fischer has obtained good results by washing out the vessels in 
the usual way with physiological salt solution, or, better still, with 
a fibrin-dissolving fluid such as a freshly filtered 8 per cent, solution 
of nitrate or sulphate of sodium and then injecting good fresh 



HISTOLOGICAL METHODS. Ifl 

milk. Fix the tissues for at least twenty-four hours in a 10 per 
cent, solution of formalin plus 2 per cent, of acetic acid. 

Cut frozen sections and stain with Scharlach R. Counterstain 
in alum-hematoxylin. Mount in glycerin. The fat can also be 
stained with osmium tetraoxid. The capillaries are outlined by 
the fat emulsion stained red or black. 

Warm Injection-masses. — 1. Berlin Blue. — Warm the 
solution of Berlin blue given above, and add it, with con- 
tinual stirring, to an equal quantity of a warm, concentrated 
solution of gelatin prepared as follows : Allow clean sheets 
of the best French gelatin to swell up for one to two hours 
at room-temperature in double the quantity of water. Then 
dissolve them by warming gently over a water-bath. Filter 
the combined solution through flannel. 

2. Carmine-gelatin Mass. — This is by all means the best 
injection-mass to use, because it is permanent, but it is very 
difficult to prepare. 

Dissolve 2 to 2.5 grams of best carmine in about 15 c.c. 
of water, to which just enough ammonia is added, drop by 
drop, to effect the solution. Filter the fluid obtained, and 
add it, with continual stirring, to a filtered warm, concen- 
trated solution of gelatin (prepared as above) over the water- 
bath. Then add acetic acid slowly until the color changes 
to a bright-red shade. The exact amount desired is when 
the solution loses its ammoniacal odor and has a peculiar 
sweetish aroma free from acid. Examined under the micro- 
scope, no granular precipitate of carmine should appear. If 
too much acetic acid has been added, so that the carmine is 
precipitated, the mass must be thrown away and a new lot 
prepared. 

Organs which have been injected with a cold mass are 
placed directly in 80 per cent, alcohol. After a few hours 
they are to be cut up into pieces that are not too small. 
After a warm injection-mass the organ or animal is placed 
first in cold water to hasten the solidification of the gelatin, 
and then transferred to 80 per cent, alcohol. Masses already 
prepared for injecting cold or warm can be obtained from 
Gruebler. 



38 PATHOLOGICAL TECHNIQUE. 

FIXING REAGENTS. 

The various reagents used for the preservation of fresh 
tissues possess the properties of penetrating, killing, fixing, 
hardening, and preserving in different degrees. Of these 
properties "fixing" is the most important, and to a certain 
extent implies or includes the others. The term " fixative " 
has been used more particularly, perhaps, for reagents which 
preserve faithfully the various changes of the nucleus in 
kaiyomitosis. In a broader sense, however, it refers to the 
faithful preservation of any tissue-element or pathological 
product, and of the chemical properties peculiar to that ele- 
ment or product. A good fixative is a reagent that pene- 
trates and kills tissues quickly, preserves the tissue-elements, 
and particularly the nuclei, faithfully in the condition in 
which they are at the moment when the reagent acts on 
them, and hardens or so affects them that they will not be 
altered by the various after-steps of dehydrating, embedding, 
staining, clearing, and mounting. Most fixatives are mix- 
tures of different reagents so combined that all the desirable 
properties may be present in as large a degree as possible. 

The choice of the proper fixing reagent for a given tissue 
is often difficult, and must depend largely on the nature of 
the pathological lesions present or suspected, and on the 
purposes for which the tissue is preserved. The best general 
fixative yet devised for faithful preservation of all kinds of 
tissues is Zenker's fluid. It is recommended above all 
others after many years of constant trial. 

Helly's modification of it is preferred by some and is 
indispensable for the preservation of certain cytoplasmic 
granules which are dissolved by the acetic acid in Zenker's 
fluid. Orth's fluid, perhaps, ranks next, but does not per- 
mit nearly so great a variety of stains to be used after it 
as Zenker's fluid. It has the advantage of costing much 
less. As a general fixative for all sorts of tissues when the 
main desire is to obtain reasonably faithful fixation for 
diagnostic purposes, formaldehyde has, to a large extent, re- 
placed alcohol. It permits about all the chemical reactions 
to be performed which are possible after alcohol fixation, and 



HISTOLOGICAL METHODS. 39 

has the additional advantage of preserving fat of all kinds, 
and especially the myelin in the sheaths of nerve-fibers. 

It is strongly advised that in all important cases tissues be 
hardened both in Zenker's fluid and in formaldehyde: in 
Zenker's fluid for general histological study, and for the 
preservation of nuclear figures, bacteria, and fibrils of all 
kinds ; in formaldehyde for the preservation of fat, myelin, 
and various substances, such as amyloid and hemosiderin, to 
which it may be desirable to apply chemical tests. For 
certain specific purposes other fixatives are sometimes re- 
quired, such as alcohol for the preservation of glycogen, pig- 
ments and sodium urate crystals, and corrosive sublimate for 
mucus. 

Tissues fixed in a solution of formaldehyde or in alcohol 
may remain as long as desirable in those fluids. Tissues 
hardened in most of the other fixatives must be transferred, 
after thorough washing in water, to alcohol for preservation. 
It is usually recommended to pass the specimens through 
graded alcohols, either through 30, 60, 90, and 96 per 
cent, or through 50, 70, and 96 per cent., allowing them to 
remain from a few hours to a day in each strength. For 
most purposes it will be found sufficient to transfer the speci- 
mens directly from water to alcohol of 70 to 80 per cent., in 
which they may remain until it is desired to imbed them. 

Alcohol extracts chrome salts from tissues hardened in solu- 
tions of them. As these salts are precipitated in the alcohol 
under the action of light, it is desirable, although by no means 
necessary, to keep all such specimens in the dark. 

It is strongly urged by some that distilled water be used in 
making all fixing solutions, and also that all fixatives be em- 
ployed at body temperature because they will then penetrate 
more quickly and the tissues will, therefore, be preserved 
more faithfully. 

Alcohol is a fair general fixative which both hardens and 
dehydrates tissues at the same time. As a fixing reagent 
formerly in much use its place is largely taken nowadays by 
formaldehyde. In its favor are several points. Bacteria, 
fibrin, various pigments, elastic fibrils, and certain cyto- 
plasmic granules stain well after it, and it is the only fixative 



40 PATHOLOGICAL TECHNIQUE. 

which preserves glycogen and allows it to be stained dif- 
ferentially. Its disadvantages are that it removes hemo- 
globin from the red blood-corpuscles, shrinks tissues more 
or less, and does not give them so good a consistence as some 
of the other fixatives. Its greatest use is as a preservative of 
tissues after they have been fixed and hardened by other 
reagents. The strength of alcohol ordinarily used in labora- 
tories is 95 to 96 per cent. Absolute alcohol is much more 
expensive. Tissues hardened in either of these strengths 
shrink a great deal. The exposed surface becomes ex- 
tremely hard, and the outer layers of the cells of tissues, like 
a rabbit's kidney, for example, are as shrunken and flat- 
tened as though dried in the air. It is only inside of this 
hard casing, where the alcohol has penetrated more slowly 
and has been somewhat diluted by the fluid of the tissue, 
that the cells are better preserved. Moreover, this ex- 
treme hardening of the surface hinders the penetration of 
the alcohol into the deeper parts. 

Tissue which is to be hardened in absolute or 95 per cent, 
alcohol should be cut into thin pieces, preferably not over 
\ cm. thick. The volume of alcohol used for hardening 
should be fifteen to twenty times as great as the specimen, 
and should be changed after three or four hours. The tissue 
should be kept in the upper part of the alcohol by means 
of absorbent cotton, or the jar may be frequently inverted 
and the alcohol thus kept of even strength. 

The advantages of strong alcohol, 95 per cent, and abso- 
lute, are that the tissue is more quickly fixed than with 
weaker strength, and that at the same time it is made quite 
hard — a quality more necessary formerly than now when tis- 
sues are so generally embedded. Tissues hardened in strong 
alcohol should later be transferred to 80 per cent, alcohol for 
preservation, or the staining properties will gradually become 
impaired. 

For general purposes it will be found better to place tissues 
at first into 80 per cent, alcohol, which should be replaced 
in two to four hours by 95 per cent, alcohol. In this way 
less shrinkage is caused and the surface of the tissues is not 
made so hard. 



HISTOLOGICAL METHODS. 4 1 

Tissues which have been fixed in Zenker's and other fluids 
should, after thorough washing in running water, be placed 
directly in 80 per cent, alcohol for further preservation. 
Change the alcohol occasionally as it becomes cloudy. 

Formaldehyde. — The gas formaldehyde (HCOH) is 
soluble in water to the extent of 40 per cent. Solutions of 
this strength are manufactured by different commercial 
houses under the names of formaline, formol, and formalose.. 
The best strength of formaldehyde to use for fixing tissues is 
a 4 per cent, solution; that is, 10 parts of the aqueous 40 per 
cent, solution, no matter what name is given to it, to 90 
parts of water. Unfortunately, formic acid gradually de- 
velops in formaldehyde and exerts an injurious action on 
tissues preserved in it. 

On this account it is advisable for most purposes to neu- 
tralize the stock solution of formaldehyde by adding car- 
bonate of calcium (powdered marble serves well) or lead 
oxide or carbonate in excess. 

For certain purposes, however, it is sometimes advisable 
to add 5 per cent, by volume of glacial acetic acid to the 
ordinary solution in order to improve its fixing properties, 
but tissues cannot be left in the mixture. They must be 
transferred after twenty-four hours to the plain formalde- 
hyde solution. 

Formaldehyde penetrates very quickly. Its hardening 
action is not understood. It does not precipitate albu- 
minous bodies, but makes them quite firm. It also hardens 
nerve-sheaths, acting toward them and red blood-corpuscles 
like the chrome salts. Formaldehyde is very useful for pre- 
serving gross specimens, because it gives them a rather 
tough, elastic consistence and preserves the normal color 
better than other hardening fluids, and also the transparency 
of many parts, such as the cornea. In general histological 
work formaldehyde is largely used now-a-days as a fixative 
in place of alcohol. 

As a fixative for specimens that are to be embedded in 
paraffin it is not recommended unless combined with other 
reagents, such as bichromate of potassium in Orth's fluid, 
because it does not appear to harden the tissue elements suf- 



42 PATHOLOGICAL TECHNIQUE. 

ficiently to enable them to resist the shrinking effects of pro- 
longed exposure to alcohol and heat in the process of em- 
bedding. In frozen sections, however, prepared by the 
method described elsewhere, this shrinkage of the tissue ele- 
ments is not apparent, probably because prolonged ex- 
posure to dehydrating, clearing, and embedding agents is 
avoided. 

The advantages of formaldehyde are that it is compara- 
tively cheap, can be obtained commercially in compact 
form, and keeps well. It fixes and hardens tissues, including 
red blood-corpuscles, quickly and well even in large pieces 
and gives them a good consistence, so that they can be cut 
easily on the freezing microtome or after embedding in 
celloidin. It permits the use of a large variety of staining 
methods. It also fixes and preserves fat so that this sub- 
stance can be easily stained in frozen sections. In addition, 
it preserves myelin, and on this account is the best prelimi- 
nary fixative of the central nervous system that we have, but 
must be followed by a chrome salt or be combined with it. 

The disadvantages of formaldehyde are that it dissolves 
glycogen and uric acid and biurate of sodium crystals, 
often changes bile concretions from a yellow to a green color, 
does not preserve iron and other pigments so well as alcohol, 
and frequently gives rise to a fine dark brown or black 
crystalline precipitate in the tissues. Two methods are 
recommended for removing the precipitate. 

A. Verocay's method: 

1. Place the sections in the following mixture for ten 
minutes: 

i per cent, aqueous solution of caustic potash, I c.c. 
80 per cent, alcohol, 100 c.c. 

2. Wash thoroughly in at least two changes of water for 
five minutes. 

3. Place in 80 per cent, alcohol for five minutes. 

4. Return again to water. 

B. Schridde's method: 

I. Place sections in the following mixture for half an hour: 

75 per cent, alcohol, 200 c.c. 

25 per cent, solution of ammonia, 1 c.c. 



HISTOLOGICAL METHODS. 43 

2. Wash thoroughly in water. 

Formaldehyde does not of itself give tissues a sufficient 
consistence so that they will stand embedding in paraffin 
without shrinking. On this account it is best combined 
with a chrome salt, as in Orth's or Helly's fluid, when this 
method of embedding is desired. 

Alcohol and Formaldehyde.— 

Alcohol (95 per cent.), 90 c.c. 

Formaldehyde (40 per cent, solution), 10 c.c. 

This combination of alcohol and formaldehyde is a most 
useful fixing mixture, especially for rapid diagnosis of routine 
surgical specimens. It fixes and dehydrates at the same 
time, and yet gives better and more faithful preservation 
than acetone, which is often employed for the same purpose. 

The following method in constant use for years in the 
Pathological Laboratory of the Boston City Hospital yields 
excellent permanent mounts. Of course, all good tissues 
are also fixed in Zenker's fluid, cut after paraffin embedding, 
and stained with eosin and methylene-blue and by any 
other method that seems desirable. 

1. Fix thin sections of tissue, 2 to 4 mm. thick, 1- 4 hours. 

2. 95 per cent, alcohol, 1-2 " 

3. Absolute alcohol, 1-2 " 

4. Absolute alcohol and ether, equal parts, 1-3 " 

5. Thin celloidin, 2-12 " 

6. Thick celloidin, a few minutes to 1 hour. 

7. Chloroform, f- 1 

8. 80 per cent, alcohol, a few minutes to 1 

9. Cut and stain in alum hematoxylin and eosin, dehy- 
drate in 95 per cent alcohol, clear in oleum origani cretici, 
and mount in xylol colophonium. 

Corrosive sublimate is a most useful fixing reagent, 
but is best employed in combination with a chrome salt, 
as in Zenker's and Helly's fluids. Its great disadvantage 
when used alone is that it causes serious shrinkage of the 
cells. A second disadvantage, which attends its use under 
all conditions, is that it gives rise to a crystalline precipi- 
tate of mercuric oxide. This precipitate can be removed 



44 PATHOLOGICAL TECHNIQUE. 

from the tissues by means of iodin, which forms a colorless 
soluble compound. 

Do not add iodin to the alcohol in which the tissues are 
preserved, because prolonged treatment with iodin exerts 
an injurious effect on the staining properties of the cells. 
Embed the tissues and cut sections without removing the 
precipitate, and then treat the sections, just before staining, 
with Lugol's solution or a I per cent, alcoholic solution of 
iodin for ten to twenty minutes, followed by alcohol to re- 
move the iodin. 

Inasmuch as prolonged action with alcohol is frequently 
necessary in order to remove the iodin, it is often better to 
use a 5 per cent, aqueous solution of sodium hyposulphite 
for this purpose instead of alcohol, because it acts almost 
instantaneously and is then itself easily removed by thor- 
ough washing in water. 

It is only fair to state that many workers prefer to add 
iodin to the alcohol in the stock jar of tissue until the color 
no longer disappears in order to remove the precipitate 
before embedding and cutting sections. 

The directions for the use of corrosive sublimate are as fol- 
lows: Use a saturated aqueous solution made with the aid 
of heat. 

The addition of 5 per cent, of glacial acetic acid is usu- 
ally advisable. 1. Harden thin pieces of tissue (2 to 5 mm.) 
for six to twenty-four hours. 2. Wash in running water 
twenty-four hours. 3. Preserve in 80 per cent, alcohol. 

Tissues hardened in corrosive stain quickly and brilliantly 
in nearly all staining solutions. It is the only fixative after 
which the Heidenhain-Biondi triple stain gives good results. 

Giemsa's Corrosive Sublimate -alcohol Fixative. — 
Saturated aqueous solution of corrosive sublimate, 2 parts; 
Absolute alcohol, 1 part. 

It requires at least forty-eight hours, and is to be renewed 
after twenty-four hours. The tissue may remain as long 
as three months in the solution without disadvantage if 
evaporation is prevented. 

This fixative is the one usually recommended for tissues 
which are to be stained by Giemsa's method, but S. B. 



HISTOLOGICAL METHODS. 45 

Wolbach has shown that excellent results, but with a re- 
versal of the color effect, may be obtained after fixation in 
Zenker's fluid, and the tissue preservation is much better. 
Zenker's Fluid.— 



Bichromate of potassium, 


2.5 grams; 


Corrosive sublimate, 


5 to 8 grams; 


Water, 


ad 100 c.c. ; 


Glacial acetic acid, 


5 cc. 



Dissolve the corrosive sublimate and the bichromate of 
potassium in the water with the aid of heat. 

Do not add the acetic acid to the stock solution, but 
only in the proper proportion to the part taken for harden- 
ing pieces of tissue, because the acid evaporates so readily, 
and also produces changes in the chrome salt. 

Zenker's fluid was originally Miiller's fluid plus 5 per cent, 
of corrosive sublimate and 5 per cent, of glacial acetic acid, 
but the sulphate of sodium is usually omitted nowadays 
because it is generally agreed that it serves no useful pur- 
pose. Personally we have always added corrosive subli- 
mate in excess (7 to 8 grams to each 100 c.c. of fluid) so as 
to have the solution saturated with it. 

Directions j or Use. — 1. Fix tissues in the solution twelve 
to twenty-four hours. 

2. Wash in running water twelve to twenty- four hours. 

3. Preserve in 80 per cent, alcohol until used. 

Tissues float at first in this solution, which penetrates 
fairly quickly. 

Zenker preparations stain slowly but beautifully in alum- 
hematoxylin. The most brilliant results, however, are ob- 
tained by staining with eosin, followed by Unna's alkaline 
methylene-blue solution. Excellent results are also obtained 
after staining in phosphotungstic-acid hematoxylin, and by 
the anilin-blue method. They bring out fibrin and various 
kinds of fibrils in addition to nuclear details. 

When sections of Zenker — fixed tissues which have been 
kept for a long time — are stained with alum-hematoxylin the 
places where the crystalline deposit was present are colored 
deep blue and thus disfigure the specimen. The only way 



46 PATHOLOGICAL TECHNIQUE. 

found so far to prevent this staining is to soak the section? 
for several weeks in acid alcohol before staining them. This 
treatment causes no injury to the tissues, but does, as a 
rule, prevent the disfiguring stains from appearing. On the 
other hand, the method will also remove certain pigments 
from the sections and, therefore, cannot always be used. 

Hellys fluid is a slight modification of Zenker's fluid: 
the glacial acetic acid is replaced by 5, occasionally 10, per 
cent, of strong formaldehyde added just before the mixture 
is used. For certain purposes, such as fixing the cytoplas- 
mic granules in the islet cells of the pancreas, the formalde- 
hyde should be carefuliy neutralized. 

Bichromate of potassium, 2.5 gr.; 

Corrosive sublimate, 5 to 8 gr. ; 

Water, 100 c.c. ; 

Formaldehyde (40 per cent, solution), 5 to 10 c.c. 

1. Fix tissues in the fluid for twelve to twenty-four hours. 

2. Wash in running water twelve to twenty-four hours. 

3. Transfer to 80 per cent, alcohol. 

Chrome Salts. — Chromic acid is rarely used nowadays 
except in Flemming's solution. The chrome salts are em- 
ployed instead, especially the bichromate of potassium, 
which enters into several well-known fixing solutions. It 
penetrates slowly and is a poor fixative of nuclear material, 
but is the best of all known hardening reagents. On this 
account tissues fixed in solutions containing it stand paraf- 
fin embedding with little or no shrinkage. Bichromate of 
potassium has been used so long in the solution known as 
Miiller's fluid that the latter solution is regarded as practi- 
cally synonymous with it. 

Miiller's Fluid.— 

Bichromate of potassium. 2 to 2.5 grams; 

Sulphate of sodium, 1 gram ; 

Water, 100 c.c. 

Harden tissues six to eight weeks. Change the fluid daily 
during the first week ; once a week thereafter. Ordinary 
tissues are then washed in running water overnight before 



HISTOLOGICAL METHODS. 47 

being placed in alcohol. Nervous tissue is transferred di- 
rectly from the fluid to the alcohol. 

This famous hardening solution is rapidly giving way to 
better fixatives. It hardens tissues slowly, evenly, and 
with little or no shrinkage, but it is a poor nuclear fixative, 
and does not encourage any great variety of stains. The 
sulphate of sodium seems to serve absolutely no function. 
For ordinary tissues it is being replaced by Zenker's, Helly's, 
and Orth's fluids, all of which fix very quickly, besides 
having all its good qualities. For nervous tissues formalde- 
hyde followed by other solutions of the chrome salts is a 
great deal quicker and better. 

Tellyesniczky has recently recommended the following- 
mixture, which has met with considerable favor, and which 
may be regarded as an improved Muller's fluid: 

Bichromate of potassium, 3 parts ; 

Water, 100 " 

Glacial acetic acid, 5 " 

Fix thin sections for one to two days; thicker sections 
longer. Wash out thoroughly in running water. Dehy- 
drate in graded alcohols. 

Orth's Fluid. — This is a general fixative consisting of 
the well-known Muller's fluid plus 4 per cent, of formal- 
dehyde : 

Bichromate of potassium, 2 to 2.5; 

Water, 100; 

Formaldehyde (40 per cent, solution), 10. 

The formaldehyde should be added only at the time of 
using, for in two days the solution becomes darker, and by 
the fourth day a crystalline deposit begins to take place. 
As fixation is ordinarily complete in three to four days, this 
deposit does not matter. The tissue should not be over 1 
cm. in thickness. Small pieces, \ to \ cm. in thickness, can 
be readily hardened in the incubator in three hours. The 
specimens should be washed thoroughly in running water 
six to twenty-four hours before placing in 80 per cent, 
alcohol. 



48 PATHOLOGICAL TECHNIQUE. 

The method is particularly recommended for mitosis, red 
blood-corpuscles, bone, and colloid material (in cystomata, 
etc.), as it gives a very good consistence to the tissues, but 
the histological detail is not so good as after Zenker's fluid. 
The addition of 5 per cent, of acetic acid would unquestion- 
ably improve it. 

Osmic Acid. — The tetroxide of osmium, commonly- 
known as osmic acid, is a fixing reagent of considerable 
value, particularly for the demonstration of fat, but pene- 
trates tissues poorly. On this account it is generally used 
in combination with other reagents some of which seem to 
increase its power of penetration. 
Flemming's Solution. — 

Osmic acid, 2 per cent, aqueous solution, 4 ; 

Chromic acid, 1 per cent, aqueous solution, 1 5 ; 

Glacial acetic acid, 1. 

I. Fix in the solution one to three days. 2. Wash in running 
water six to twenty-four hours. 3. Alcohol, 80 per cent. 

It is best to keep the osmic acid in a 2 per cent, solution 
and the chromic acid in a 1 per cent, solution. The mixture 
can then be quickly made up fresh at the time it is needed. 
The best stains after hardening in Flemming are Babes' saf- 
ranin, aniline-methyl-violet, and carbol-fuchsin. 

Pieces of tissue for hardening in Flemming's solution 
should not be over 2 mm. in thickness, because it has very 
slight penetrating properties. 

Marchi's Fluid.— 

Miiller's fluid, 2 parts; 

Osmic acid, 1 per cent, aqueous solution, 1 part. 

Place small pieces of tissue in the mixture for five to eight 
days, wash thoroughly in running water, and harden in 
alcohol. For its application to degenerated nerve-fibers 
see page 147. 

Hermann's Solution. — 

Osmic acid, 2 per cent, aqueous solution, 4; 

Platinic chlorid, 1 per cent, aqueous solution, 15 ; 

Glacial acetic acid. I. 



HISTOLOGICAL METHODS. 49 

This modification of Flemming's solution is perhaps an 
even better fixative than the model on which it is based, 
but is more expensive. It should be employed in the same 
manner. 

Pianese's Solution. — 

Chlorid of platinum and sodium, 1 per cent. 

aqueous solution (platinic), 1 5 c.c. ; 

Chromic acid, \ per cent, aqueous solution, 5 " 
Osmic acid, 2 per cent, aqueous solution, 5 " 

Formic acid, C. P., I drop. 

Fix small pieces of tissue, not over 2 mm. thick, in the solu- 
tion for thirty-six hours. Wash in running water for twelve 
hours, then 80 per cent, alcohol. Stain paraffin sections by 
Pianese's special methods (see p. 80). 

This fixative and the special staining methods are particu- 
larly recommended for the study of karyomitosis and of the 
so-called cancer bodies. 

Boiling". — Boiling precipitates the soluble albumin in 
tissues as a granular material which can be readily recog- 
nized. The method is used particularly for the demonstra- 
tion of albumin in renal diseases and in edema of the lungs. 
By means of boiling the quickest permanent mounts of 
tissues can be obtained. The method is not advocated on 
account of the shrinkage caused by the heat, but will some- 
times be found useful. Occasionally 10 per cent, or even 
40 per cent, formaldehyde is employed instead of water. 

Small pieces of tissue not over 1.5 cm. in diameter should 
be dropped into the boiling water for one-half to two min- 
utes; cool quickly in cold water, and make frozen sections, 
or put into 80 per cent, alcohol. Any stain may be used, 
methylene-blue will be found excellent. 

DECALCIFICATION. 

Tissues which are to be decalcified should be sawn with 
a fine hair-saw into thin slices, so that they will decalcify 
quickly. It is usually desirable to saw the tissue into pieces 



50 PATHOLOGICAL TECHNIQUE. 

of proper size for embedding in celloidin. Very dense bone 
ought not to be over 2 or 3 mm, thick; softer tissues do not 
need to be thinner than 4 to 6 mm. In cutting sections after 
decalcifying and embedding it is necessary to throw away the 
first half-dozen sections or so, because the tissue is so lacer- 
ated to a slight depth by the movement of small fragments 
of bone in the process of sawing as to be useless for micro- 
scopic purposes. The extent of the decalcification may be 
tested at any time by thrusting a needle into the tissue, but 
it is best to avoid such a test because, of course, it tends 
to produce injury to the tissue. 

The following steps in the decalcification of tissues must 
be carefully borne in mind. 

1. The tissues must first be thoroughly hardened. The 
three most useful reagents for this purpose are alcohol and 
Zenker's and Orth's fluids. After the two latter reagents the 
tissues must have been washed thoroughly in water and 
placed in alcohol for at least twenty-four hours. They will 
then be ready for decalcification. 

2. The decalcifying fluid must be used in large amounts, 
and, if necessary, be frequently changed. Decalcification 
should never be prolonged beyond four days if possible ; 
twenty-four to forty-eight hours are better. 

3. After decalcification the tissues must be thoroughly 
washed in running water for twenty-four hours to get rid of 
every trace of the acid. 

4. The tissues finally must be hardened again in alcohol. 
Of the various agents used for decalcifying bone, nitric, 

hydrochloric, chromic, picric, trichloracetic acids, etc., the 
most important is nitric acid. It acts quickly, without swell- 
ing the tissues or attacking injuriously the tissue-elements, 
and does not interfere to any marked degree with any sub- 
sequent staining process. Red blood-corpuscles will be found 
uninjured in tissues hardened in Zenker's fluid even after re- 
maining four days in 5 per cent, nitric acid. This acid is 
used in dilute solution alone or in combination with phloro- 
glucin. 
Directions for Using Nitric Acid.— 1. Decalcify in 



HISTOLOGICAL METHODS. 5 1 

large quantities of a 5 per cent, aqueous solution of nitric 
acid, changing the solution every day for one to four days. 
2. Wash twenty-four hours in running water to remove 
every trace of acid. 3. Harden in 80 per cent., and then 
95 per cent, alcohol. Embed in celloidin. According to 
Schaffer, it is best to transfer the tissue directly from the 
nitric acid to a 5 per cent, solution of alum for twenty-four 
hours before placing in running water, so as to avoid any 
possibility of the tissue swelling, but this step hardly seems 
necessary. 

Schridde recommends highly the following strong solu- 
tion for rapid decalcification: 

Formaldehyde (strong solution), ioc.c; 

Distilled water, 90 c. a; 

Nitric acid, 20 c.c. 

Use at body temperature; two to three hours are usually 
sufficient, but tissues may remain in the solution for twenty- 
four to forty-eight hours. Wash for twelve to twenty-four 
hours in running water and then transfer to 80 per cent, 
alcohol. 

Phloroglucin and Nitric Acid. — Phloroglucin is not 
a decalcifying agent, but is added to nitric acid to protect the 
tissues while allowing a stronger solution of the acid to be 
used than would otherwise be possible. The solution is 
prepared by dissolving 1 gram of phloroglucin in 10 c.c. of 
nitric acid. Solution takes place quickly, with generation of 
considerable heat. The fluid is reddish brown at first, but 
becomes light yellow in the course of twenty-four hours. 
Dilute with 100 c.c. of a 10 per cent, solution of nitric acid. 
This gives nearly a 20 per cent, solution of nitric acid. The 
process of decalcification in this fluid is extremely rapid ; a 
few hours only, as a rule, are required. It is not advisable 
to dilute the solution by the simple addition of water, but 
by the use of less acid, because the phloroglucin must be 
present to the amount of 1 per cent, or it will not protect 
the tissues so well. 



S 2 PATHOLOGICAL TECHNIQUE. 

The following slower-acting solution may be found useful : 
Phloroglucin, I • 

Nitric acid, 5 j 

Alcohol, 70 • 

Water, 30. 

A rather deep single stain with alum-hem atoxylin (either 
aqueous solution or Delafield's) will usually be found to give 
the best results with tissues decalcified with nitric acid. It 
is very important to leave the sections after staining in a 
large dish of water overnight, otherwise the stain will not 
be so sharp and clear. 

Sulphurous Acid.— A saturated solution— about 5 per 
cent. — is used. It works very quickly and causes little 
swelling. The tissues should be carefully washed out in 
running "water as after nitric acid. The stock solution 
rapidly grows weak through evaporation if the bottle in 
which it comes is not kept tightly corked. 

Trichloracetic Acid.— A 5 per cent, solution of this 
acid has lately been recommended for the decalcification of 
bone and teeth. It acts more slowly than nitric acid, and 
seems to possess no advantages over it. Tissues must be 
washed out in running water, as after nitric acid. 

FROZEN SECTIONS. 

In making frozen sections with a simple microtome such 
as described on page 20 the following directions may be 
helpful. It should be emphasized that satisfactory sections 
are much more easily made with the more complicated 
machines, especially those with automatic feed mechanism. 

The knife must be sharp and free from nicks. It must 
have a chisel edge, as shown in Fig. 2. It should be 
sharpened by grinding on a hone, and afterward by thor- 
oughly stropping on a razor-strop. Frequent stropping is 
just as necessary as in the case of the ordinary microtome 
knife. 

In cutting, grasp the knife by the thick wooden handle so 
that the end presses against the ball of the thumb and the 
palm of the hand, while the dorsum of the hand is upper- 



HISTOLOGICAL METHODS. 53 

most; then, with the wrist flexed and held against the chest, 
apply the edge of the knife to the glass ways of the micro- 
tome in such a manner that the edge, bevel side downward, 
is at right angles to the direction of the ways and the long 
axis of the knife at an angle of 45 degrees to their surface ; 
now, holding the knife and wrist rigidly in the positions just 
indicated, push the cutting-edge quickly forward along the 
ways through the specimen by moving the body forward 
from the waist, in the mean while pressing the cutting-edge 
steadily downward upon the ways with constant force. Thus 
a strong constant downward pressure of the edge upon the 
ways is maintained, and at the same time great steadiness 
and power are given to the cutting stroke, which are condi- 
tions that are very important for obtaining thin sections. 
With the fingers of the other hand manipulating the wheel 
of the microtome screw, a number of sections should be cut 
in quick succession in the manner indicated without chang- 
ing the angle of the knife or the position of the hand and 
wrist above described, the edge of the knife on the back- 
ward movement being lifted from the ways only enough to 
clear the cut surface of the specimen. The sections will 
usually adhere to the knife, and a number of them may be 
allowed to collect thereon. They are removed from the 
knife by immersing it in water, in which they will float and 
flatten out, no matter how much wrinkled and compressed 
upon the knife they have been. The cutting of a number 
of sections in quick succession without pausing to remove 
each section from the knife seems to be necessary for obtain- 
ing the thinnest sections. 

The consistence of the frozen tissue is important. The 
specimen immediately after freezing will usually be too hard 
to cut without yielding sections that break over the edge of 
the knife, and are, therefore, to be rejected. If this happens, 
wait a few seconds and thereafter cut a section or two at 
short intervals until the specimen is found to have a con- 
sistence yielding satisfactory sections, whereupon a number 
of sections should be cut in quick succession as above 
described. 



54 PATHOLOGICAL TECHNIQUE. 

The piece of tissue from which the sections are to be cut 
should be not thicker than 5 mm., and a little water should 
be placed under it on the freezing box to bind it thereon. 

Before staining, the section should be fastened to the slide 
to avoid distortion and facilitate handling. The best way 
of doing this is as follows: 

Coat the slide with a thick layer of Mayer's albumin 
fixative and float the section on to it, spreading smoothly. 
Next wipe away most of the fluid from around the section 
and press it on to the slide with smooth blotting paper. 
Then, without allowing the section to dry out, cover it with 
a mixture of equal parts of aniline oil and clove oil, and 
immediately rinse off the mixture with 95 per cent, alcohol. 
After immersing in water to remove the alcohol the section 
is ready for staining and mounting. 

In spreading the section on the slide too long immersion 
of the slide in the water may wash off the fixative and the 
section will not stick. This very rarely happens after a 
little practice. 

Goodpasture recommends the following method of stain- 
ing frozen sections of tissues fixed in formaldehyde or in 
Orth's or Helly's fluid: 

1. Place sections in his acid polychrome methylene-blue 
solution for one minute or longer; they will not overstain. 

2. Wash and mount in water. 

Nuclei deep purple; connective tissue a bright rose red. 
The method will not work with frozen sections of fresh tissues. 

Wright's Embedding Method for Frozen Sec- 
tions. — To obtain frozen sections of small fragments of 
tissue, such as curettings of the uterus, so that they may 
be stained and mounted as a single section, embedding in 
gelatin may be employed. 

The embedding mass consists of a 10 per cent, solution 
of sheet gelatin in distilled water, with which, while warm 
and fluid, | of 1 per cent, carbolic acid has been mixed. 

It keeps well. 

To embed, the mass is liquefied in a water-bath, a small 
pool poured either on a smooth hard surface or in a 
paper box, and the pieces of tissue arranged therein. 



HISTOLOGICAL METHODS. 55 

After about half an hour or longer a "block" containing 
the tissue is cut out of the solidified mass for sectioning. 

It is important that the section be fastened to the slide 
as described above before staining. Further, overheating 
and consequent breaking down of the gelatin, as well as 
drying of the block, should be avoided. 

CELLOIDIN AND PARAFFIN EMBEDDING PROC- 
ESSES. 

Sections of hardened tissues can be cut with a razor by 
hand, or with a microtome knife after fastening the specimen 
in the microtome clamp either directly or between pieces of 
amyloid liver. Fair sections of firm tissues can often be ob- 
tained in this way. Thinner sections can be got by means 
of the freezing microtome, but these methods are all open 
to the objection that unless the tissue is very cohesive, por- 
tions of it are likely to fall out of the sections. 

The best results would, therefore, naturally be expected 
from some embedding process, employing a substance to in- 
filtrate the tissues thoroughly and to hold the different parts 
in proper relative position even in the thinnest sections. 

The two substances in common use for this purpose are 
celloidin and paraffin. Each has its advantages and disad- 
vantages. Neither can be employed in pathological his- 
tology to the exclusion of the other. Paraffin affords the 
thinner sections, but they must be small if the best results 
are desired, and cannot be properly handled except when 
fastened to the slide. Hard tissues, like muscle, and tissues 
of varying consistency, like skin, are cut with considerable 
difficulty by the paraffin method. Staining is rather simpler 
than after embedding in celloidin. 

On the other hand, tissues of almost any consistency or 
size can be cut by the celloidin method, which is also capa- 
ble of furnishing very thin sections. 

Both methods of embedding should be learned and used. 
Celloidin sections are especially good for general work, for 
studying the extent and relations of pathological processes, 
and for much of the finer histological work. Paraffin sec- 



56 PATHOLOGICAL TECHNIQUE. 

tions are better for the finest details of processes — for special 
work on special tissues. 

Celloidin. — Schering's celloidin has in the past been the 
best preparation of gun-cotton (pyroxylin) to use; It keeps 
well, dissolves somewhat slowly, and gives a fairly trans- 
parent embedding mass, which is firm and tough, so that 
very thin sections can be cut. Mallinckrodt's Purified Py- 
roxylin, recently put on the market, seems to give equally 
good results. 

Embedding in Celloidin. — The process consists in 
soaking the tissues for twenty-four hours to a number of 
days in two different solutions of celloidin. The two solu- 
tions are spoken of as thin and thick celloidin. To make 
thick celloidin 30 grams of the dry celloidin are dissolved 
in 200 to 250 c.c. of a mixture of equal parts of ether and 
absolute alcohol. Diluted with an equal amount of the 
ether-and-alcohol mixture, it forms thin celloidin. 

The steps of the imbedding process are as follows : Pieces 
of tissue which have been properly fixed and finally pre- 
served in 80 per cent, alcohol are first to be cut up with in- 
telligence. They should rarely be over 2 to 4 mm. thick ; 
for most purposes 2 mm. will be found sufficient. Pieces of 
this thickness will furnish a hundred sections or more, will 
embed more quickly than larger masses, and will be more rigid 
when mounted on a block. They should never be broader 
or longer than is necessary to show the whole of the process 
under study. Very thin celloidin sections cannot usually be 
obtained with tissues over i| to 2 cm. square, and smaller 
dimensions are preferable. Beginners usually imbed larger 
pieces than are necessary. 

The trimmed pieces of tissue are first hardened and de- 
hydrated for twenty-four hours in 95 per cent, alcohol, fol- 
lowed by twenty-four hours in absolute alcohol ; then soaked 
in equal parts of absolute alcohol and ether for the same 
length of time to prepare them for the thin celloidin. In the 
latter they remain at least twenty-four hours, preferably for 
a number of days, if at all thick, for in this solution occurs 
most of the infiltration with celloidin. Finally, the pieces are 
soaked twenty-four hours or more in the thick celloidin. 



HISTOLOGICAL METHODS. 57 

They are then mounted on blocks of vulcanized fiber, 
placed in chloroform for one or two hours, and then trans- 
ferred to 80 per cent, alcohol. 

Briefly summed up, the steps of embedding in celloidin are 
as follows: 

1. 95 per cent, alcohol, twenty-four hours. 

2. Absolute alcohol, twenty-four hours. 

3. Ether and absolute alcohol, equal parts, twenty-four 
hours. 

4. Thin celloidin, twenty-four hours to one or more weeks. 

5. Thick celloidin, twenty-four hours to one or more weeks. 

6. Mount on blocks of vulcanized fiber. 

7. Harden celloidin in chloroform for one or two hours, 
followed by 80 per cent, alcohol. 

Instead of mounting directly from the thick celloidin, it is 
often advisable to allow the celloidin to evaporate until a 
firm mass is obtained. This is particularly true when very 
loose tissues are to be embedded. 

The simplest method is to place the pieces of tissue, which 
have been soaking in thick celloidin, in proper position in a 
glass dish and pour thick celloidin over them. The dish is 
then covered, but not too tightly, and the ether is allowed to 
evaporate for one or more days until the proper consistency 
of celloidin is reached, so that it can be cut out in blocks 
enclosing the specimens. If the ether evaporates too 
rapidly, place a large dish or a bell-jar over the covered 
dish. Mount the blocks, after they have been cut out and 
trimmed, by dipping the bases in thick celloidin and then 
pressing them on to blocks of vulcanized fiber. 

Place them in chloroform for one or two hours and then 
transfer to 80 per cent, alcohol. 

After the celloidin mounts have been in 80 per cent, 
alcohol for one to several hours, the celloidin is of the proper 
consistence for cutting. It is best to take a sharp knife or 
an old razor and trim the top of the celloidin down to where 
the first good section of the specimen can be cut; this will 
save considerable wear on the microtome knife. 

In cutting, the microtome knife should be fastened very 
obliquely, so that as much as the edge of the knife as pos- 



58 PATHOLOGICAL TECHNIQUE. 

sible shall be used in making each section. The surface of 
the knife should be kept well wet with 80 per cent, alcohol, 
preferably from an overhanging drop-bottle. 

If the sections curl, as often happens when they are thin, 
they are best flattened by unrolling them on to the surface 
of the knife with a camel's-hair brush just before the last 
edge of celloidin is cut through, as this serves to keep them 
fixed in place during the process. This method can be used 
when the simple transferring of sections from alcohol to 
water is not sufficient to uncurl them. 

Celloidin sections can be stained by nearly all methods, 
without the necessity of removing the celloidin. When 
necessary, however, the celloidin is readily removed by 
placing the sections from absolute alcohol in oil of cloves 
or in the alcohol-and-ether mixture for five or ten minutes, 
and then passing them back through absolute into ordinary 
alcohol. 

To Attach Celloidin Sections to the Slide. — A celloidin sec- 
tion can be fairly well attached to a slide by transferring it 
from water to a slide freshly washed in alcohol and dried 
with a cloth. The section is then to be firmly blotted with 
filter-paper so as to apply it closely to the slide and to re- 
move all wrinkles. It should not be allowed to dry. A 
section treated in this way will ordinarily stand considerable 
manipulation without becoming loose. 

Celloidin sections can be more securely attached by trans- 
ferring them from 95 per cent, alcohol to clean slides and 
pouring over them ether-vapor from a bottle half full of 
ether. With a little practice sections can be fastened in a 
few seconds. Follow slowly along the edge of the celloidin, 
and the frills in it will soften down. Then wash the specimen 
with 80 per cent, alcohol to harden the celloidin. 

Another excellent method is that described for fixing 
frozen sections to the slide (see page 54). 

Paraffin. — Excellent paraffin, melting at I25°F. (51. 6° C), 
can be obtained, when bought in large quantities, for about 
eight cents a pound, from the regular dealers in paraffin, 
and can be used at all seasons of the year. (E. F. King 



HISTOLOGICAL METHODS. 59 

& Co., Boston, can be recommended.) We have never found 
the more expensive sorts recommended by dealers in labor- 
atory supplies necessary. 

Embedding in Paraffin. — Paraffin embedding is par- 
ticularly useful when very thin sections are desired. To ob- 
tain the best results the pieces of tissue should be small, soft, 
and of uniform consistence. In pathological work it is much 
better to cut the sections and to stain them after they are 
fastened to the slide than to stain in the mass beforehand, 
because then a variety of stains may be used. A complete 
or perfect series is not so important as in embryology, but 
with a little care can be obtained. 

The first step in the preparation of hardened tissues for 
the paraffin bath is to cut them into small, thin, square or 
rectangular pieces, not over I cm. square, perhaps, for the 
best results, and not over 2 to 3 mm. thick. It should be 
stated, however, that with proper skill, a heavy, sharp knife, 
and a rigid microtome very thin paraffin sections can be ob- 
tained with tissues measuring 4X3 cm. The pieces of 
tissue are then thoroughly dehydrated by soaking first in 95 
per cent, and then in absolute alcohol. From alcohol they 
are put in some substance, such as chloroform or oil of 
cedar, which has the property of mixing with alcohol and 
of dissolving paraffin. From the chloroform they are trans- 
ferred to a saturated solution of paraffin in chloroform, and 
then passed through two separate baths of the melted par- 
affin to get rid of every trace of the chloroform. If oil of 
cedar is used, the specimens are transferred directly from it 
into the melted paraffin, or they may be placed first for 
half an hour or so in chloroform to get rid of the oil of 
cedar. This procedure enables one to make use, for certain 
dense tissues, such as the skin, of the better penetrating powers 
of the oil, and yet avoid carrying it into the paraffin bath. 

One advantage of the chloroform method is that the dura- 
tion in the hot paraffin, the objectionable feature of the par- 
affin method, is shortened, because the tissues are already 
somewhat infiltrated with paraffin. Another advantage is 



6o 



PA THOL O GICAL TECHNIQ UE, 



that the paraffin bath purines itself, because the chloroform 
rapidly evaporates. When oil of cedar is used, the paraffin 
must be renewed frequently. 

Benzene (benzol) is preferred by some workers to chloro- 
form or xylol and can be highly recommended. It clears 
quickly, renders the tissues more transparent than the 
other reagents do, and evaporates rapidly from the paraf- 
fin bath, 

The methods of embedding in paraffin are briefly stated as 
follows : 

Method No. i. 

1. 95 per cent, alcohol, 6-24 hours. 

2. Absolute alcohol, 6-24 " 

3. Chloroform, 6-24 " 

4. Chloroform saturated with paraffin, 6-24 " 

5. Paraffin bath, two changes, 2- 4 " 

6. Embed and cool quickly in cold water. 



Method No. 2. 

1. 95 per cent, alcohol, 

2. Absolute alcohol, 

3. Oil of cedar, two changes, 

4. Paraffin, three changes, 

until no odor of oil of cedar. 
5 Embed and cool quickly in cold water. 



6-24 hours. 
6-24 " 
6-24 " 



\- 2 hours. 
15-30 minutes. 
30-90 



Method No. j. 

1. Acetone, 

2. Benzene, 

3. Paraffin, 

This method is recommended when there is great haste. 
We are not sure that it does not shrink the tissue more than 
the other methods. The quantity of acetone used should be 
at least twenty-five times the volume of the tissue. With 
larger amounts of tissue the acetone should be changed after 
thirty minutes or an hour, and a longer exposure to the 
acetone and paraffin may be necessary. 



HISTOLOGICAL METHODS. 6 1 

Method No. 4. 

The following method is recommended for certain brittle 
tissues, such as guinea-pigs' livers, which are difficult to 
section after fixation in Zenker's fluid: 

1. Transfer tissue .from 80 per cent, alcohol to 95 per 
cent, alcohol for one and a half hours. 

2. Absolute alcohol, one and a half hours. 

3. Oil of cedar, two changes, forty-eight hours. 

4. Blot: Xylol five minutes, two changes, two and a half 
minutes each. 

5. Paraffin oven, four changes in all, two hours. 

For embedding paraffin specimens metallic boxes can be 
used, or forms made round or square from strips of sheet 
lead or tin. Many prefer paper boxes, which can be made 
easily of any size desired from stiff writing-paper. 

Melted paraffin is poured into the paper box to the depth 
of about I cm. The pieces of tissue are then placed in the 
box with that side down from which sections are preferred. 
When all the pieces are arranged in order with about half 
a centimeter or more between them, the box is placed on the 
surface of a large dish of cold water, on which it floats, so 
that the paraffin may cool quickly without crystallizing. 
Sometimes it is advisable to set the paper box with the 
specimens in it in the paraffin oven for a short while, so as 
to get rid of any bubbles carried in by the specimens. After 
the paraffin has hardened, the paper is removed and the par- 
affin is divided up according to the pieces in it. One of the 
blocks is then fastened to the object-holder by heating the 
latter in a flame until it will just melt the paraffin when the 
block is held in proper position against it. The holder is 
then quickly cooled in cold water. 

The upper surface of the paraffin should now be shaved 
down to the specimen. The four sides are to be carefully 
trimmed; the upper and lower surfaces should be parallel 
and not cut too close to the specimen, otherwise the sections 
will not adhere to each other ; the lateral surfaces should, 
as a rule, be cut close to the tissue, especially if very thin 
sections are desired, because if a rim of paraffin is left it is 



62 PATHOLOGICAL TECHNIQUE. 

likely to cause wrinkling of the sections. The holder is 
finally carefully adjusted in the paraffin microtome. 

To get good sections which will adhere to each other and 
form a ribbon the temperature of the room must be regulated 
to suit the degree of hardness of the paraffin used. An open 
window will often make all the difference needed to obtain 
good results. The harder the paraffin the warmer the room 
must be. The temperature can be raised by burning a Bun- 
sen flame near the microtome or lowered by the presence of 
a lump of ice. It will often be found advantageous to dip 
the holder and paraffin block into ice water just before cut- 
ting sections, or to rest a small bag containing cracked ice 
on the block and knife for a few minutes just before cutting. 

The ribbons of sections as cut, usually a slideful, are 
laid on the surface of a large dish of warm water at about 
44 C, and if necessary gently stretched so as to remove 
all wrinkles. Paint the surface of a slide with a thin layer 
of Mayer's glycerin-albumin mixture, wipe off all excess with 
a towel so that only a faint layer is left, dip the slide under 
the sections, arrange them in order, lift the slide, and drain 
off the water. The slide is then placed in a slanting position 
until dry, when it is put in the incubator for two to twelve 
hours at a temperature of about 54 C. This process attaches 
the sections firmly to the slide. 

To get rid of the paraffin in the sections they are treated 
with two or three changes of xylol, and then with absolute 
followed by 95 per cent, alcohol. 

If for any reason the celloidin-and-oil-of-cloves mixture is 
used for attaching the sections to the slide, the paraffin is 
removed by means of xylol, followed by origanum or berga- 
mot oil, and finally by 95 per cent, alcohol, because absolute 
alcohol will dissolve the celloidin. 

Mayer's glycerin-albumin mixture for attaching 
paraffin sections to slides is composed of equal parts of the 
white of egg and of glycerin. The mixture should be thor- 
oughly beaten and then filtered, or after standing for some 
time can be decanted. Add 1 per cent, of sodium salicylate 
to prevent decomposition. Egg-albumin is dissolved by 
acids and alkalies, so that when such reagents are to be 



HISTOLOGICAL METHODS. 63 

used the sections are best attached to the slide by some 
other substance. For this purpose Schallibaum 's solution, 
of celloidin 1 part in 3 or 4 parts of oil of cloves, is often 
useful. Cover the slide with a thin layer of the solution. 
Arrange the sections in order on the slide and place it in 
the thermostat at 54 to 6o° C. for several hours, or heat 
for a few seconds to half a minute over the flame until the 
oil of cloves runs together in drops. After cooling, remove 
the paraffin with xylol, pass through origanum oil to 95 per 
cent, alcohol, and proceed as with other paraffin sections. 

SERIAL SECTIONS. 

By the Celloidin Method. — i . With a little care perfect 
serial sections can be made by the following method, and each 
slide of sections can be stained in whatever way seems best. 
The specimen is embedded, mounted on vulcanized fiber, and 
hardened in 80 per cent, alcohol in the usual way. In cut- 
ting moisten the microtome knife with 95 per cent, alcohol. 
As the sections are cut they are drawn up on the surface of 
the knife and arranged in regular order by means of a camel's- 
hair brush until a slideful is ready. They are then drawn on 
a clean and numbered slide held against the back of the 
knife. After being carefully arranged the sections are fast- 
ened to the slide by means of ether-vapor (see p. 58) poured 
over them from a half-full bottle. Care must be taken that 
every edge of the celloidin is fully softened down. The slides 
are then placed in a jar of 80 per cent, alcohol to be stained 
at leisure. 

2. Another method, often convenient where the stain is of 
little importance, is as follows : The tissue is stained, in bulk, 
in alum-cochineal or some other staining fluid that will pene- 
trate, and then imbedded in celloidin in the usual way. After 
being mounted on vulcanized fiber the specimen is hardened 
in chloroform instead of in 80 per cent, alcohol. From the 
chloroform the specimen is transferred to oil of thyme. 
After it is thoroughly penetrated by the latter it is ready to 
be cut. The knife is to be moistened with oil of thyme. 
The sections as cut are arranged on the knife, and then trans- 



64 PATHOLOGICAL TECHNIQUE. 

ferred to slides placed against the back of the knife. The 
slides covered with sections can be placed under a bell-jar as 
fast as they are ready until all are cut, because the oil of 
thyme evaporates slowly. Balsam and cover-slips can be 
added after the cutting is finished. 

3. Darkschewitsch has recently proposed a comparatively 
simple method for preparing a series of celloidin sections. 
Take a glass cylinder without a neck, of about the diameter 
of the specimen to be cut. Prepare a series of circles of 
filter-paper cut of a size just to fit the bottle, number in 
order, and wet them with alcohol. Each section is removed 
from the microtome knife by pressing one of the paper 
circles upon it and drawing it off. The paper is then in-, 
verted so that the section is uppermost, and deposited in 
proper order in the bottle, where the series forms a column, 
each section resting upon a numbered paper. The sections 
can be kept indefinitely by filling the bottles with 80 per 
cent, alcohol. When ready to stain, the alcohol is poured 
off, the sections washed with water if necessary, and then the 
staining solution poured into the bottle. Other reagents 
are used in the same manner, or sections can be treated with 
the reagents in flat plates, as they do not readily slip off the 
papers. 

4. Weigert's method for a series of celloidin sections was 
designed especially for the nervous system and is rather 
complicated. The process depends on transferring the sec- 
tions as cut to narrow strips of tissue-paper. To do this 
each section as cut is arranged in proper position close to 
the edge of the knife. Then a strip of tissue-paper twice as 
wide as the section is gently placed upon it, and the sections 
withdrawn from the knife. The success of the process 
depends on having but little alcohol on the knife, other- 
wise the specimen will not stick. Each specimen is placed 
on the paper to the right of the last one. The strips of 
paper when full are kept moist by being placed with the 
specimens uppermost on a moist surface composed of a layer 
of -blotting-paper wet with alcohol, covered with a sheet of 
tissue-paper, and lying in a shallow dish. 



HISTOLOGICAL METHODS. 65 

When all the sections have been cut, each strip of them 
is taken in turn and coated on both sides with a thin film of 
celloidin in the following way : A strip of sections with the 
specimens below is first pressed gently down upon the sur- 
face of a slide covered with a thin layer of celloidin. This 
fastens the sections and the paper can be removed. Then a 
thin coat of celloidin is poured over the sections and the 
slide is placed on its edge to drain. When the surface of 
the celloidin is dry, the strips can be marked by a fine brush 
dipped in methylene-blue. As soon as the slides are placed 
in the staining solution the celloidin peels off, taking the 
specimens with it. Later, the strips of specimens can be 
divided as desired. On account of their thickness they 
should be cleared, after dehydrating in 95 per cent, alco- 
hol, in a mixture of xylol 3, carbolic-acid crystals 1. 
5. F. H. Verhoeff recommends this method: 
In cutting the sections, the knife is not carried entirely 
through the celloidin block, but an uncut edge, about 3 mm. 
wide, is left each time. After twenty or more sections are 
cut in this way the knife is carried all the way through, thus 
producing a little book of sections. It is probably most 
convenient to keep each book in a separate bottle; but no 
difficulty is usually experienced in determining the proper 
order after the sections are mounted. Another way to keep 
them in order is to string them on a silk thread through 
their uncut margins. In beginning a new book a wider 
margin should be left for the first one or two sections, as 
otherwise the sections may not adhere, or the first section 
may be cut at double thickness. Each book is stained in 
the same manner as a single section, except that it is best to 
use slow-acting stains, so that the staining will be uniform 
throughout. The individual sections are not separated until 
the book is in alcohol preliminary to clearing. Then each 
section is either torn off with forceps, or the book is taken 
up on cigarette paper and the uncut margin removed with 
scissors. Each section in order is then removed, cleared 
quickly in oil of origanum, and placed on a slide. 

5 



66 PATHOLOGICAL TECHNIQUE. 

6. Suzuki recommends spreading the sections out on a 
slide or glass plate, blotting the celloidin at one corner of the 
section, and marking the number of the section on it with a 
certain Japanese or Chinese ink by means of a fine-pointed 
brush. It is said that the solid India ink freshly rubbed up 
with a little water is satisfactory for the purpose. The sec- 
tions are placed in 80 per cent, alcohol after marking. 

By the Paraffin Method. — To obtain serial sections by 
the paraffin method it is only necessary to avoid losing any of 
the sections from the ribbon as ordinarily cut. Perhaps the 
easiest and safest way is to cut long ribbons, a yard or more 
in length, and to place them on sheets of paper in proper 
order. They can then readily be divided by means of needles 
into short series of any desired number of sections, and 
fastened to numbered slides by means of albumin fixative. 

STAINING SOLUTIONS. 

Hematoxylin and Hematein Stains.— The active 
coloring agent in most hematoxylin stains is hematein, 
which is gradually formed in the ordinary solutions from 
the hematoxylin by oxidation, a process occupying a number 
of days or weeks and spoken of as " ripening." The selec- 
tive staining power of alum-hematoxylin solutions is due to 
the combination of this hematein with aluminium. The 
resulting blue-colored solution is precipitated in the tissues 
(chiefly in the nuclei) by certain organic and inorganic salts 
there present, as, for instance, phosphates. 

Mayer and Unna have shown that it is possible to oxidize 
and to ripen in an instant a solution of alum and hema- 
toxylin by adding to it a little peroxid of hydrogen neutral- 
ized by a crystal of soda. 

By employing hematein or its ammonium salt, instead of 
hematoxylin, Mayer has been able to obtain immediately 
ripened solutions which compare fairly favorably with old 
and well-known solutions prepared from hematoxylin by the 
slow process of ripening. They do not stain any better, 
however, and it is doubtful if, for the present at least, they 
become generally acceptecj. 



HISTOLOGICAL METHODS. 67 

Most solutions of alum and hematoxylin are not stable. 
A continuous chemical change is the formation from hema- 
toxylin, by oxidation, of hematein, which, uniting with the 
alum, gives a bluish or purplish solution. The degree of 
blueness depends largely on the freshness of the alum. As 
the solution becomes older free sulphuric acid is gradually 
formed from the alum, causing the solution to lose its bluish 
or purplish tint and to become reddish. A third chemical 
change is the continuous formation of a precipitate due to 
the further oxidation of the hematein, in consequence of 
which it is always necessary to filter alum-hematoxylin solu- 
tions just before they are used. 

More alum than is needed to combine chemically with the 
hematoxylin is always added to the solution, for the reason 
that it acts as a differential decolorizer, limiting the stain largely 
to the nuclei of the cells. As alum-hematoxylin solutions be- 
come older they stain more quickly, but also more diffusely. 
This diffuseness of staining can be counteracted by adding 
enough alum-water to make the stain precise again. A good 
alum-hematoxylin solution ought not to stain the celloidin 
in which the section is imbedded. If the celloidin stains 
more or less deeply, it shows that the solution requires more 
alum. 

Aqueous Alum-hematoxylin Solution. — 

Hematoxylin crystals, I ; 

Saturated aqueous solution of ammonia alum, 100; 
Water, 300 ; 

Thymol, a crystal. 

The hematoxylin crystals are dissolved in a little water by 
the aid of heat. The combined solution is exposed to the 
light in a bottle lightly stoppered with a plug of cotton. 
The solution will be ripened sufficiently for use in about 
ten days, after which time it should be kept in a tightly 
stoppered bottle. The solution is very easily prepared, 
gives beautiful results, and will keep at its best for two to 
three months. The proportions of alum and of hematox- 
ylin are the same as in Delafield's solution. For Zenker 



68 PATHOLOGICAL TECHNIQUE. 

preparations, which stain very slowly, it will be found more 
convenient to omit the 300 c.c. of water in the preceding 
formula. 

Mallory's Instantaneous Alum Hematoxylin. — 
Hematoxylin, 1 gram; 

Ammonia alum, 10 grains; 

Water, 400 c.c; 

0.25 per cent, aqueous solution of per- 
manganate of potassium, 10 
Thymol, a crystal. 

Pulverize the hematoxylin in a mortar and dissolve it 
and the alum in the water with the aid of heat. After the 
solution is cool add the permanganate of potassium and 
then the thymol. The stain is ready to use at once. If 
ammonium hematein is used instead of hematoxylin, take 
but 5 c.c. of the permanganate of potassium solution. 

If a saturated solution of alum is kept on hand it may be 
more convenient to employ 100 c.c. of it and 300 c.c. 
of water in making up the staining fluid. 

As the solution ripens with age and tends to stain diffusely 
add a little saturated alum solution to render its action 
more precise 

Delafield's Hematoxylin. — 

Hematoxylin crystals, 4 grams ; 

Alcohol, 95 per cent., 25 c.c. ; 

Saturated aqueous solution of ammonia 

alum, 400 " 

Add the hematoxylin dissolved in the alcohol to the alum 
solution, and expose the mixture in an unstoppered bottle 
to the light and air for three to four days. 
Filter, and add — 

Glycerin, 100 c.c ; 

Alcohol, 95 per cent, 100 " 

Allow the solution to stand in the light until the color is 
sufficiently dark, then filter and keep in a tightly-stoppered 



HISTOLOGICAL METHODS. 69 

bottle. The solution keeps well and is extremely powerful. 
So long as it is good the solution has a purplish tinge. 

It would seem advisable, both in this solution and in Ehr- 
lich's, to combine the alum, hematoxylin, and the water, and 
to ripen the solution for two or three weeks before adding 
the other ingredients which have a tendency to prevent oxi- 
dation. A fully ripened solution would then be obtained 
more quickly and surely. 

Harris's Hematoxylin. — 

Hematoxylin, 1 gram ; 

Alcohol, 10 c.c. ; 

(Dissolve the hematoxylin in the alcohol.) 
Alum (ammonium or potassium), 20 grams ; 

Distilled water, 200 c.c. 

Dissolve the alum in the water by the aid of heat, and add 
the hematoxylin solution. Bring the mixture to a boil as 
rapidly as possible, and then add a half gram of mercuric 
oxide. The solution at once assumes a dark purple color. 
As soon as this occurs, remove the vessel containing the 
solution from the flame, and cool by plunging at once into a 
basin of cool water. As soon as cool, the solution is ready 
for staining. This solution keeps for years in a well- stop- 
pered bottle (Harris). 

The addition of 4 per cent, of glacial acetic acid increases 
the precision of the nuclear staining. 

This stain is especially adapted for sections fixed in 
Zenker's fluid. 

Mayer's Hemalum. — 

Hematein, or its ammonia salt, 1 gram ; 

90 per cent, alcohol, 50 c.c. ; 

Alum, 50 grams ; 

Water, 1000 c.c; 

Thymol, a crystal. 

Dissolve the hematein or its ammonia salt in the alcohol by 
the aid of heat, and add it to the alum dissolved in the water. 



*]Q PATHOLOGICAL TECHNIQUE. 

The solution can be diluted with 20 parts of water or of 
weak alum solution. 

Mayer's Acid Hemalum is prepared by adding 2 per 
cent, of glacial acetic acid to the above solution. The acid 
stain is more precise than the alkaline. 

Mayer's Glycerin-alum-hematein Solution. — According 
to Mayer's latest investigations, glycerin is the only reliable 
preservative of hematein solutions. Unfortunately, it slows 
the staining power to a considerable extent and makes the 
stain less precise. He recommends the following solution 
for its keeping properties : 

Hematein, 0.4 grams 

(dissolve by rubbing up in a few drops of glycerin) ; 



Alum, 


5 grams ; 


Glycerin, 


30 c.c. ; 


Water, 


70 " 


Weigert's Alcohol Hematoxylin. — 




Hematoxylin crystals, 


10 grams ; 


Alcohol (absolute or 95 per cent), 


90 c.c. 



The solution ripens in a week or two to a brown color, and 
keeps perfectly for a long time. It is used only in the 
Weigert stain for myelin sheaths, for which purpose it is 
diluted at the time of using with water and combined with 
carbonate of lithium (see page 137). 

Mallory's Phosphomolybdic Acid Hematoxylin. — 

Hematoxylin crystals, 1.75 grams ; 

Phosphomolybdic acid crystals, I gram ; 

Water, 200 c.c. 

The hematoxylin will dissolve almost immediately if pow- 
dered, or it may be dissolved in water by the aid of heat. 
The solution must be exposed to the light in a bottle plugged 
with cotton for five to six weeks before it is fully ripened. 
It will keep for several months, and can be used over and 
over. It is employed for staining the nervous system and 
connective tissue. 



HISTOLOGICAL METHODS. J I 

This stain was useful after fixation in Muller's fluid. It 
does not give very satisfactory results after formaldehyde 
followed by Weigert's quick mordants. 

Mallory's Phosphotung-stic Acid Hematoxylin. — 
Hematein ammonium, o. I gram ; 

Water, ioo c.c. ; 

Phosphotungstic acid crystals (Merck), 2 grams. 

Dissolve the hematein in a little water by the aid of heat, 
and add it after it is cool to the rest of the solution ; no 
preservative is required. If the solution stains weakly at first, 
it may be ripened by the addition of 5 c.c. of a J per cent, 
aqueous solution of permanganate of potassium, or it may be 
allowed to stand for a few weeks until it ripens spontaneously. 

Hematoxylin may be used instead of hematein ammonium, 
but requires 10 c.c. of the permanganate solution to ripen it. 

This staining solution will be found particularly useful for 
the demonstration of fibrin and of neuroglia, fibroglia, and 
myoglia fibrils. It also brings out with great sharpness and 
faithfulness of detail the structures in mitosis, including the 
spindles and centrosomes. 

Carmine Stains. — The active staining principle in car- 
mine solutions is carminic acid. In cochineal carminic acid 
is combined with an alkaline base. Carmine itself is a com- 
mercial compound containing carminic acid combined with 
aluminum and calcium. Carminic acid itself does not stain, 
but it forms compounds with certain metals, mainly with the 
aluminum contained in alum, which have selective staining 
properties. 

All of the alkaline and acid solutions made with carmine 
owe their staining properties to carminic acid combined with 
the aluminum, and perhaps also to the calcium contained in 
the carmine. 

Alum Carmine. — 

Carmine, 2 grams ; 

Alum, 5 " 

Water, 100 c.c. 



J 2 PATHOLOGICAL TECHNIQUE. 

Boil twenty minutes, adding enough water to make up for 
that lost by evaporation. When cool, filter and add a 
crystal of thymol to prevent the growth of mould. 

Alum Cochineal. — 

Powdered cochineal, 6 grams ; 

Ammonia alum, 6 " 

Water, ioo c.c. 

Boil for half an hour ; add water to make up for that lost 
by evaporation. Filter and add a crystal of thymol. 
Mayer's Alcoholic Carmine (Paracarmine). — 

Carminic acid, i.o gram ; 

Chlorid of aluminium, 0.5 " 

Chlorid of calcium, 4.0 grams ; 

70 per cent, alcohol, 100.0 c.c. 

Dissolve cold or warm ; allow to settle, then filter. After 
staining, wash out in 70 per cent, alcohol to which is added 
2.5 per cent, glacial acetic acid if a more purely nuclear 
stain is desired. 



Orth's Lithium Carmine. — 






Carmine, 




2.5 to 5 grams; 


Saturated aqueous solution of 


car- 




bonate of lithium, 




100 c.c. ; 


Thymol, 




a crystal. 



The carmine dissolves at once in the cold solution. When 
used as a counter-stain for bacteria in the Gram-Weigert 
method this solution should be carefully filtered, because 
organisms occasionally grow in it and may give rise to con- 
fusion in the stained preparations. 

Neutral Carmine. — Dissolve, without heating, 1 gram of 
best carmine in 50 c.c. of distilled water plus 5 c.c. of strong 
aqua ammoniae. Expose the fluid in an open dish until it 
no longer smells ammoniacal (about three days) ; then filter 
and put away in a bottle for future use. The odor of the 
solution will soon become bad, but the staining properties 
will remain unaffected. 



HISTOLOGICAL ME TIIODS. 73 

Aniline Dyes.^We have been dependent in the past 
on Germany for practically all our aniline dyes. For bac- 
teriologic and pathologic work those obtained from Grubler 
were the most reliable. They were special brands which 
had been tested by men of authority. Small quantities 
of them are still on the market. Now, owing to the war, we 
have to take what we can get. Many dye manufactories 
have started up in this country, but their products have not 
yet been tested and classified for our kind of work, and it 
will probably take some time before the best varieties have 
been sorted out. In the meantime we shall have to do the 
best we can. 

Aniline dyes come in the form of a powder or as crystals, 
and most of them keep well in that condition. Methylene- 
blue for one, however, seems to be an exception. After the 
original package has been opened for a short while the dye is 
said to lose in intensity of staining power. It is well to keep 
on hand saturated alcoholic solutions of certain of the dyes, 
because they keep well in that form, and are ready for use 
when a saturated alcoholic solution is wanted. This is 
particularly true of methylene-blue, fuchsin, and methyl- 
violet. 

Aniline dyes are derived from either aniline or toluidin, or 
from both together. They may be regarded as salts having 
basic or acid properties. The basic colors stain cell-nuclei, 
including bacteria, for which they show a marked affinity. 
The acid colors stain diffusely. The basic dyes most com- 
monly employed in pathological histology are methylene- 
blue, fuchsin, methyl-violet, and safranin. Of the acid 
colors, eosin, picric acid, and acid fuchsin are most in use. 

As a rule, every aniline dye has one or more standard 
solutions which are used largely to the exclusion of others, 
for the reason that, being required for certain purposes, they 
are kept in stock. As they are thus always at hand, they 
are used where simple solutions might be used. For in- 
stance, Loffler's methylene-blue solution is often used, be- 
cause ready and convenient, when a simple aqueous solution 
would do as well. 

In the following pages we have arranged under each dye 
the solutions of it most in use : 



74 PATHOLOGICAL TECHNIQUE. 

Methylene-blue.— i. Saturated solution in 95 per cent, 
or absolute alcohol. A stock solution to be used in making- 
other solutions. It can be used as a stain by adding 1 part 
to 9 parts of water. 

2. Aqueous solutions of various strengths are often used, 
and can be made up as needed. 

3. Loffler's Methylene-blue Solution. — 

Saturated alcoholic solution of methylene-blue, 30 c.c. ; 
Solution of caustic potash in water, 1 : 10,000, 100 " 

This is one of the most useful of the aniline staining solu- 
tions, and will keep for a long time without losing much in 
staining power. 

4. Kuhne's Methylene-blue Solution. — 

Saturated alcoholic solution of methylene-blue, 10; 
5 per cent, carbolic-acid water, 90. 

This is a stronger staining solution than Loffler's, but the 
resulting stain does not seem so sharp and clear. 

5. Gabbets Methylene-blue Solution. — 
Methylene-blue, 2 ; 
Sulphuric acid, 25 ; 
Water, 75. 

It is used as a decolorizer and contrast-stain for tubercle 
bacilli. 

6. Unnds Alkaline Methylene-blue Solution. — The strongly 
alkaline solution of methylene-blue recommended by Unna 
for staining plasma-cells has been found extremely valuable 
as a general stain in connection with eosin, which should be 
used first. The solution should be diluted I : 10, or 1 : 5, 
for staining ; it stains better after ripening for a week or two : 

Methylene-blue, I ; 

Carbonate of potassium, 1 ; 

Water, I OO. 
(For method of using see page no.) 

7. Unnds Polychrome Methylene-blue Solution. — The poly- 
chrome methylene-blue solution, much used by Unna in 
various staining methods, is an old alkaline solution of 



HISTOLOGICAL METHODS. 75 

methylene-blue, of which the one on page 74 is the orig- 
inal formula, in which, in consequence of oxidation, methyl- 
violet and methylene-red have formed. Months are re- 
quired for the process of oxidation to take place. The 
ripened solution may be obtained from Griibler. 

8. Goodpasture 's Acid Polychrome Methylene-blue Solu- 
tion. — 

Methylene-blue (Koch f. bac), 1 gram; 

Potassium carbonate, 1 " 

Distilled water, 400 c.c. 

Dissolve the ingredients thoroughly and boil in a flask for 
thirty minutes. The methylene-blue will be polychromed 
and most of it precipitated. When the solution is cool add 
3 c.c. of glacial acetic acid. Shake thoroughly until the 
precipitate is dissolved and then boil gently for five minutes 
or until the solution is concentrated to a volume of 200 c.c. 
Cool it in tap-water. It is ready for use immediately, may 
be used over and over, does not precipitate, and keeps 
indefinitely. 

9. SahWs Borax Methylene-blue Solution. — 
Saturated aqueous solution of methylene-blue, 24 c.c. ; 
5 per cent, solution of borax, 16 c.c; 
Water, 40 c.c; 

Mix, let stand a day, and filter. 

Fuchsin. — 1. Saturated alcoholic solution to be kept in 
stock. 

2. Ziehl-Neelsons Carbol- fuchsin. — 

Saturated alcoholic solution of fuchsin, 10 c.c; 
5 per cent, carbolic-acid water, 90 " 

Carbolic acid water is made by shaking together 5 c.c. of 
melted carbolic-acid crystals and 95 c.c. of water. The 
solution should be filtered. 

This solution is very powerful, stains quickly, keeps well, 
and can be employed for a variety of purposes. 

3. Verhoeff's Carbol-fuchsin Solution. — 

Carbolic acid, melted, 25 c.c; 

Absolute alcohol, 50 " 

Fuchsin, 2 grams. 



76 PATHOLOGICAL TECHNIQUE. 

Combine the ingredients and place over night in an incu- 
bator to ensure complete solution ; cool and filter. This 
stock solution of carbol-fuchsin, unlike the dilute aqueous 
solution which slowly deteriorates, is permanent and does 
not even require to be filtered again. 

For use in staining coverslip preparations, add two drops 
of this stock solution to eight drops of distilled water. 
When larger quantities of staining solution are required, the 
dilution is made in the proportion of I c.c. of the stock solu- 
tion to 6 c.c. of distilled water. 

4. Aniline -fuchsin. — 

Saturated alcoholic solution of fuchsin, 16 c.c. ; 
Aniline-water, 84 " 

Methyl- violet. — 1. Aqueous solutions of various strengths, 
J to 2 per cent., keep well and are used for staining nuclei, 
bacteria, and amyloid. 

2. Methyl-violet can be used instead of gentian-violet in 
Ehrlich's solution. Weigert recommends two permanent 
stock solutions by means of which the aniline methyl- 
violet solution can be made up easily when wanted. 

Solution 1. — Absolute alcohol, 33; 

Aniline, 9 ; 

Methyl-violet in excess. 

Solution 2. Saturated aqueous solution of methyl-violet. 
The staining solution consists of — 

Solution 1, 1 ; 

Solution 2, 9. 

This mixture will keep at the most for fourteen days. 

3. For staining neuroglia-fibers Weigert employs a satu- 
rated solution made with the aid of heat in 70-80 per cent, 
alcohol. 

Gentian-violet. — This dye is not a definite chemical sub- 
stance, but a mixture of crystal-violet, methyl-violet, and 
dextrin. It is better to discard it entirely, and to use methyl- 
violet instead in the staining solutions given ; they are cited 
here as originally given only because they are classical. 



HISTOLOGICAL METHODS. *]J 

1. Saturated alcoholic solution to be kept in stock. 

2. EhrlicJis Aniline- gentian-violet. — 

Saturated alcoholic solution of gentian-violet, 16 c.c. ; 
Aniline-water, 84 

Aniline-water (aniline oil water) is made by shaking to- 
gether 5 parts of aniline with 95 parts of water, and filtering 
the resulting milky fluid. It should come through perfectly 
clear. During the first few hours after the solution is made 
considerable precipitation takes place, so that it is best not to 
use it for twenty-four hours. After about ten days it begins 
to lose its staining power. (See under Methyl-violet, page 

76.) 

Zenker recommends a solution without alcohol : Dissolve 
the gentian-violet directly in the aniline-water. The color is 
said to be less easily removed from tissues when this solu- 
tion is used. 

3. Stirling's Solution of Gentian-violet. — 

Gentian-violet, 5 grams; 

Alcohol, 10 c.c. ; 
Aniline, 2 " 

Water, 88 " 

This solution is said to keep remarkably well. 

4. Carbol- gentian Violet. — 

Saturated alcoholic solution of gentian-violet, 10 c.c; 
5 per cent, carbolic-acid water, 90 " 

Safranin. — Two of the many preparations by this name 
have been found especially useful : 

1. Safranin O soluble in water. 

2. Safranin soluble in alcohol. 

The three following solutions of safranin can be thoroughly 
recommended : 

1. Saturated aqueous solution of "safranin O soluble in 
water" (to be made with the aid of heat). 

2. A mixture of equal parts of — 

A saturated aqueous solution of " safranin O soluble 

in water." 
A saturated alcoholic solution of " safranin soluble in 

alcohol." 



78 PATHOLOGICAL TECHNIQUE. 

Babes' Aniline Safranin. — 

2 per cent, aniline water, ioo; 

"Safranin O soluble in water," in excess. 

Saturate the solution by heating it in a flask set in hot water 
to 60-80 C. ; filter. 

This solution is extremely powerful, stains almost in- 
stantly, and will keep about two months. 

Bismarck Brown. — The most common solutions are the 
following : 

1. A 1 per cent, aqueous solution. 

2. A saturated aqueous solution made by boiling (3-4 per 
cent.). 

3. A saturated solution in 40 per cent, alcohol (2-2J per 
cent.). 

Unlike other aniline colors, Bismarck brown will keep in 
glycerin mounts and can be fixed in nuclei by acid alcohol. 
The stain is not used so much as formerly, except as a con- 
trast stain in Gram's method and for photographic purposes. 
Other basic stains less frequently used, and then generally 
in aqueous solutions, are dahlia, methyl-green, iodin-green, 
and thionin. 

Diffuse Stains. — 1 . Eosin is sold in two forms — as " eosin 
soluble in water," and as " eosin soluble in alcohol." The 
first is to be preferred, because a greater degree of differen- 
tiation in stain can be obtained with it. Keep on hand a 
saturated aqueous solution, to which a crystal of thymol has 
been added, and dilute with water as needed. The strength 
of solution to be used varies somewhat with the tissue and 
the reagent in which it has been fixed, but generally lies be- 
tween y 1 ^ and f per cent, when the eosin is used after a hem- 
atoxylin stain. These dilute solutions should contain 25 per 
cent, of alcohol, otherwise they will not keep well. When 
eosin is employed before an aniline dye, such as methylene- 
blue, a 5 per cent, or even a saturated solution should be 
taken. Solutions of eosin should always be filtered immedi- 
ately before use. 

2. Picric Acid. — Saturated alcoholic and aqueous solu- 
tions should be kept in stock, to be diluted as needed. 



HISTOLOGICAL METHODS. 79 

3. Acid Puchsin. — Aqueous solutions of various strengths 
are used. It is advisable to keep on hand a 5 per cent 
solution and to dilute it to the strength required. A crystal 
of thymol should be kept in the solution because otherwise 
molds readily grow in it. 

Altmann's Aniline Acid Fuchsin Solution. — 

Acid fuchsin, 20. grams ; 

Aniline water, 100. c.c. 

4. Van Gieson's Picro-fuchsin Solution. — This valuable 
solution was originally made by adding to a saturated 
aqueous solution of picric acid enough of a saturated 
aqueous solution of acid fuchsin to give to the fluid a deep 
garnet-red color, and for certain purposes, as in staining 
after Zenker's fluid, this strong solution is to be preferred. 
Freeborn has recently given more precise directions for 
making up the solution according to the purpose for which 
it is to be used. 

For Comiective Tissue. — (Seepage in). 
1 per cent, aqueous solution of acid fuchsin, 5 c.c. ; 
Saturated aqueous solution of picric acid, 100 " 

For the Nervous System. — (See page 120). 

I per cent aqueous solution of acid fuchsin, 1 5 c.c. ; 

Saturated aqueous solution of picric acid, 50 " 

Water, 50 " 

Picro-nigTosin (Martinotti). — Dissolve picric acid and 
nigrosin to saturation in 70 per cent, alcohol. 

Combination Stains. — Biondi-Heidenhain Staining- 
Solution. — 

Saturated aqueous solution of orange G, 100; 

Saturated aqueous solution of acid fuchsin or 

rubin S, 20; 

Saturated aqueous solution of methyl-green, 50. 
(About 20 gm. rubin S., 8 gm. orange G., and 8 gm. 
methyl-green; dissolve in 100 c.c. of water.) 

Make up the separate solutions and let them stand for seve- 
ral days with excess of coloring matter (shaking the bottles 



So PATHOLOGICAL TECHNIQUE. 

occasionally) until they are saturated. Then mix the solu- 
tions. For staining, dilute the combined solution with water 
I : 60 to 1 : 100. 

The following- tests are used for finding out if the proper 
combination has been obtained: The addition of acetic acid 
should make the solution redder ; a drop of the solution on 
filter-paper should make a blue spot with green in the center 
and orange at the periphery. If a red zone appears outside 
of the orange, then too much acid fuchsin is present. 

Pianese's Staining Solutions and Staining Meth- 
ods. — The following stains, devised by Pianese, are recom- 
mended by him particularly for the study of cancer, but will 
be found useful in many lines of histological investigation. 
The first two were used by him for tissues hardened in cor- 
rosive sublimate or in Zenker's fluid ; the others, only after 
his special fixative (given on page 49). The methods are 
intended for paraffin sections : 

1. Carmine and Picro-nigrosin. — 1. Stain in neutral or 
lithium carmine. 

2. Decolorize in acid alcohol. 

3. Wash in water. 

4. Absolute alcohol. 

5. Aniline-gentian-violet, ten minutes. 

6. Iodin solution, two to three minutes. 

7. Absolute alcohol, so long as any color is discharged. 

8. Saturated aqueous solution of picric acid and of nigro- 
sin, five minutes. 

9. Decolorize in a 1 per cent, alcoholic solution of oxalic 
acid. 

10. Water, several minutes. 

11. Absolute alcohol. 

12. Oil of bergamot. 

13. Balsam. 

Nuclei, red ; cell-protoplasm, light olive-green ; connec- 
tive tissue, dark olive-green ; elastic fibers, bluish ; bacteria 
and blastomycetes, violet. 

II. Methylene-blue and Eosin in Borax Solution. — 
Keep three solutions on hand : 



HISTOLOGICAL METHODS. 8 1 

(a) Saturated solution of methylene-blue in a saturated 
aqueous solution of borax. 

(&) \ per cent solution of " bluish eosin " in 70 per cent, 
alcohol. 

{c) Saturated aqueous solution of borax. 

For use mix together 2 parts of the filtered solution a, 1 
of b, and 2 of c. The different steps of the staining pro- 
cess are as follows : 

1. Absolute alcohol. 

2. Staining solution, ten to twenty minutes. 

3. Decolorize in a 1 per cent, solution of acetic acid. 

4. Wash in water. 

5. Absolute alcohol. 

6. Xylol. 

7. Xylol balsam. 

Nuclei, blue ; red blood-globules, cell-protoplasm, granules 
of eosinophiles, connective tissue, etc., rose-red. 

III. a. Malachite-green, Acid Fuchsin, and Nigrosin. — 

Malachite-green, 1. gram; 

Acid fuchsin, .4 " 

Nigrosin, .1 " 

Water, 50 c.c.; 

Alcohol saturated with acetate of copper, 50 " 

1. Absolute alcohol. 

2. Stain in 20 drops of above solution diluted with 10 c.c. 
of distilled water for twenty-four hours. 

3. Decolorize in a \ per cent, aqueous solution of oxalic 
acid. 

4. Wash in water. 

5. Absolute alcohol. . 

6. Xylol balsam. 

Resting nuclei, light red; protoplasm, reddish yellow. In 
the karyokinetic figures, nuclein green ; fibrillar of the achro- 
matic spindle and of the mitoma, bright red; centrosome 
and polar bodies, red ; the rest of the cell-body, a reddish- 
yellow color. 
6 



82 PATHOLOGICAL TECHNIQUE. 

III. b. Malachite-green, Acid Fuchsin, and Martius 
Yellow. — 

Malachite-green, .5 gram ; 

Acid fuchsin, .1 " 

Martius yellow, .01 " 

Distilled water, 150 ex.; 

Alcohol, 96 per cent., 50 " 

1. Stain in the solution without diluting, half an hour. 

2. Absolute alcohol. 

3. Xylol. 

4. Xylol balsam. 

Nuclei of resting and dividing cells, green ; cell-cytoplasm, 
connective tissue, etc., rose-colored ; " cancer-bodies," mainly 
red, but in masses of them some are red and some green. 

IV. Acid Fuchsin and Picro-nigrosin. — 
Saturated alcoholic solution of acid fuchsin, 6 drops ; 
Martinotti's picro-nigrosin, 8 " 
Distilled water, 10 c.c. 

1. 70 per cent, alcohol. 

2. Stain in the solution six hours. 

3. Decolorize in dilute acetic acid. 

4. Absolute alcohol. 

5. Xylol. 

6. Xylol balsam. 

Resting nuclei, red ; nuclein of karyokinetic figures, yellow : 
cell-protoplasm, dark olive-green ; " cancer-bodies," mainly 
olive-gray, but some or portions of them may be ruby-red. 

V. Light Green (Lichtgrun) and Hematoxylin. — 
Ehrlich's acid hematoxylin, 15 c.c. 
Saturated solution of Lichtgrun in 70 per cent. 

alcohol, 5 " 

Distilled water, 15 " 

1. Distilled water. 

2. Stain in above mixture half an hour. 

3. Wash thoroughly in several waters. 

4. Alcohol. 



HISTOLOGICAL METHODS. 83 

5. Oil of Bergamot. 

6. Balsam. 

Nuclei, green ; " cancer-bodies " take the hematoxylin stain. 

VI. Acid Puchsin and Hematoxylin. — 

Ehrlich's acid hematoxylin, 15 c.c. 

1 per cent, solution of acid fuchsin in 70 per 

cent, alcohol, 15 " 

Distilled water, 1 5 " 

Stain as in V. 
Nuclei, red ; cytoplasm, brick-red ; " cancer-bodies " take the 
hematoxylin stain. 

Orcein, a vegetable dye obtained from certain tinctorial 
lichens, is used mainly for staining elastic fibers. It is 
soluble in alcohol, and is employed either in a neutral or 
acid (HC1) alcoholic solution. 

Iodin is the oldest of the histological stains, but is now 
but little used for that purpose, except in staining amyloid 

The tincture of iodin, a saturated solution in alcohol, is 
used for getting rid of the precipitate of mercury formed in 
tissues fixed in corrosive sublimate or in Zenker's fluid. 

I,llgol'S solution, a solution of iodin in water containing 
iodid of potash, is of varying strength. Iodin in this form 
is much used as a test for starch, amyloid, glycogen, and 
corpora amylacea. In Gram's stain and its modifications iodin 
produces some chemical change in the coloring material em- 
ployed, in consequence of which, when appropriate decolor- 
izers are used, the stain remains fast in certain structures, 
while from others it is easily entirely extracted. 

The strength originally employed by Gram for his stain- 
ing method was — 

Iodin, I gram ; 

Iodid of potash, 2 grams ; 

Water, 300 c.c. 

Weigert in his modification of this method employed a 
stronger solution : 

Iodin, I g ram . 

Iodid of potash, 2 grams ; 

Water, IO o c.c. 



84 PATHOLOGICAL TECHNIQUE. 

Recently he has recommended the following strength both 
for fibrin and for neuroglia-fibers : 

Iodid of potash, c grams \ , .,, . ,. 

r ' J ^ > saturated with 10dm. 

Water, ioo c.c. J 

The only difference in the action of the various solutions 
probably is that the strong solution acts practically instan- 
taneously, while the weaker solutions require some little time. 



5TAINING riETHODS. 

The purpose of staining is to render prominent the differ- 
ent tissue-elements, so that they may be readily recognized 
and studied. The constant tendency now-a-days is toward 
selective or differential staining methods, by which but one 
tissue-element will be colored to the exclusion of all others, 
or at least of, any element that might be confused with it 
morphologically. These selective stains, which really are 
micro-chemical .color reactions, enable us to differentiate 
from each other with ease and accuracy cellular and inter- 
cellular elements, or pathological products which otherwise 
look alike. 

The list given on page 85 does not pretend to be either 
complete or perfect in arrangement, but will give some idea of 
the various elements which we wish to stain. Those for 
which we now possess more or less perfect differential stains 
are printed in italics. 

The simplest selective stain is, of course, that for nuclei, 
and it can be obtained with a great variety of staining re- 
agents. The most difficult element to stain differentially, 
although it can be done under certain conditions with a fair 
amount of success, is probably the axis-cylinder and its ter- 
minal processes. 

Tissue-elements and pathological products differ from 
each other, not only .in form and consistency, but also in 
chemical properties. While perfect preservation of form is 
sufficient to distinguish certain cells or elements from each 
other — as, for instance, polymorphonuclear leucocytes from 



Cell. 



Intercellular 
substances. 



Pathological 
products. 



Nucleus. 



Nucleolus. 
Resting nucleus. 
Linin. 

)i . Do not stain by Gram. 
2. Stain by Gram. 
3. Stain by tubercle-bacillus method. 
Nucleus of Ameba coli. 
Centrosome and polar bodies. 
Mast cell. 

Plasma-cell of Unna. 
Parietal cells, stomach. 
A and B islet cells, pancreas. 
Zymogen granules, intestine, pan- 
creas. 



Granules. 



(Five kinds of gran- 
ules described by 
Ehrlich. 
^ NissVs granules in ganglion-cells. 
Dendritic processes of ganglion-cells. 
Axis-cylinder and terminal processes. 
Contractile elements of striated muscle-cell. 
Myoglia fibrils. 
Red blood-corpuscles. 
Blood- platelets and megakaryocytes. 
Cilia of bacteria. 

Certain dots or lines in ependymal cells. 
Cuticle. { So-called cilia in certain renal cells. 
Bile-capillaries. 

Cement substance of epithelial and endothelial cells. 

Ground substance of connective tissue. 

Connective-tissue {collagen) fibrils, and reticulum. 

Fibroglia fibrils. 

Epithelial fibrils. 

Mucous connective tissue; mucin. 

Elastic fibers. 

Intercellular substances of cartilage. 

Ground substance of bone. 

Myelin. 

Neuroglia- fibrils. 

Clubs of actinomycetes. 

Capsules of bacteria. 

Fibrin. 

Mucin. 

Amyloid. 

Glycogen. 

Hyalin. 

Colloid. 

Keratohyalin. 

Eleadin. 



Calcium 

Hyaline substances 

Fat. 

Hemosiderin. 

Hematoidin. 

Hemoglobin. 



85 



86 PATHOLOGICAL TECHNIQUE. 

lymphoid cells — differentiation based on micro-chemical tests 
is always to be preferred when possible. A few of the tests 
employed are colorless, like the precipitation of mucin by 
acetic acid. Certain tests, like the methylene-blue or gold 
stain for axis-cylinders, can be applied to fresh tissues only. 

Others, like the various amyloid reactions, can be obtained 
with fresh or hardened tissues. Most of the micro-chemical 
reactions, however, can be employed only with tissues which 
have been properly preserved. It is exceedingly important, 
therefore, that a tissue-element be so fixed and hardened that 
its peculiar chemical properties be preserved intact, otherwise 
a differential stain for it is impossible. Each tissue-element 
is a law unto itself. For example, the peculiar chemical 
properties of red blood-corpuscles depend on the presence in 
them of hemoglobin. As a differential stain of the red 
blood-corpuscles depends on fixing this substance in them.it 
is necessary to find out the chemical properties of hemo- 
globin, such as the fact that it is soluble in water or dilute 
alcohol, but not in salt solution, and that it is fixed in the 
red blood-corpuscles by heat, absolute alcohol and ether 
equal parts, corrosive sublimate, formaldehyde, bichromate 
of potassium, etc. 

While differential stains depend in part on the chemical 
properties of the tissue-elements, they also depend to a cer- 
tain extent on the chemical properties of the staining re- 
agents and the decolorizers used. 

Some of the tissue-elements can be stained differentially in 
a number of ways, sometimes after one fixing agent, some- 
times after another. The simplest differential stains are those 
where certain tissue-elements stain directly in a given solu- 
tion after they have been properly fixed. Good examples 
are — Ehrlich's triple stain for certain cytoplasmic granules in 
leucocytes, and the direct stain for elastic fibers with an acid 
alcoholic solution of orcein. 

Other differential stains depend on the property of certain 
elements to hold colors they have once taken up when 
treated with decolorizers. The best example of this is the 
tubercle bacillus, which holds certain stains through various 



HISTOLOGICAL METHODS. 87 

acids or aniline hydrochlorate, followed by alcohol, and, if 
necessary, by a contrast-stain. 

Still another varied group of elements (certain bacteria, 
fibrin, neuroglia-fibers, etc.) depends for a differential stain in 
part on changes produced in methyl-violet by iodin, in part 
on the decolorizer employed for extracting the coloring 
reagent. 

Although the steps of the various staining methods differ 
considerably, they may be roughly arranged in the following 
order : 

1. Staining. 

2. Differentiating. 

3. Decolorizing. 

4. Dehydrating- 

5. Clearing. 

6. Mounting. 

Very often two or more of the steps are combined in one, 
as when aniline oil is used for decolorizing, dehydrating, and 
clearing sections stained for certain bacteria. Sometimes the 
staining process occupies more than one step, as in Weigert's 
myelin-sheath stain. In alum-hematoxylin the differentiating 
reagent, the excess of alum, is combined with the stain ; in 
Gram's method the differentiating reagent, iodin, forms a step 
by itself. 

NUCLEAR STAINS. 

For general histological work no stain is so useful or can 
be so highly recommended as the eosin-methylene-blue stain 
after fixation in Zenker's fluid. It brings out nuclei and 
nuclear figures with great sharpness, while at the same time 
it stains the cytoplasm of certain cells so that they are easily 
distinguished from other cells. Next in point of general 
usefulness is phosphotungstic acid hematoxylin, owing to the 
sharpness with which it stains nuclei and centrosomes, and 
especially nuclear figures, including the spindle. At the 
same time it demonstrates certain fibrils which other methods 
fail to show. 



88 PATHOLOGICAL TECHNIQUE. 

For class-room work alum hematoxylin, with eosin as a 
contrast stain, holds its own as the best general stain for cel- 
loidin sections after a variety of fixatives, but especially after 
Zenker's fluid. 

Of the carmine stains, lithium carmine, followed by picric 
acid, will be found the most brilliant, generally useful, and 
permanent, but is useless after Zenker fixation. 

Safranin gives, perhaps, the most permanent stain of any 
of the basic aniline dyes, and confines itself very sharply to 
the nuclei. It is much used after certain fixing reagents, such 
as Flemming's and Hermann's solutions. The Heidenhain- 
Biondi triple stain is useful after fixation in corrosive subli- 
mate, but cannot be employed with celloidin sections, so that 
its field is limited. The other aniline dyes are used on 
occasion or for some definite purpose, but not so generally 
as those mentioned above. 

A good alum-hematoxylin solution should have a bluish 
or purplish color, and should stain celloidin very faintly or 
not at all. 

Aqueous Alum-hematoxylin; Mallory's Instan- 
taneous Alum-hematoxylin; Delafield's Alum- 
hematoxylin; Harris's Alum-hematoxylin (see 
pages 67-69). 

1. Stain in one of the above solutions two, five, or thirty 
minutes, or sometimes even longer. Sections of Zenker 
fixed tissue usually require at least one hour. 

2. Wash in several changes of water, and then leave sec- 
tions, if possible, for several hours or over night in a large 
dish of water ; or better still, wash in running tap water for 
ten to thirty minutes. 

3. Contrast-stain, usually an aqueous solution of eosin, 
Yq to \ per cent, for one to five minutes. 

4. Alcohol, 95 per cent, two or three changes to dehydrate 
and to remove excess of contrast-stain. 

5. Clear in oleum origani cretici or in Dunham's oils-of- 
cloves-and-thyme mixture. 

6. Xylol colophonium or balsam. 

The more customary method of using Delafield's alum- 



HISTOLOGICAL METHODS. 89 

hematoxylin solution is to filter a few drops of it into a dish 
of water and to stain sections for a long time, even over 
night, with the very dilute solution thus obtained. It is 
sometimes advisable to use the aqueous solution in the same 
way. 

Mayers Hemalum (see page 69).— i. Stain three to 
five minutes or longer. 

2. Wash out in 1 per cent, alum solution until the stain is 
precise. 

3., Wash thoroughly in several changes of water. 

4. Alcohol, 95 per cent. 

5. Oleum origani cretici. 

6. Xylol balsam. 

The staining is rather diffuse, so that it has to be washed 
out to some extent with alum-water. Mayer's acid hemalum 
is more precise, and usually does not need to be decolorized, 
so that the second step can be omitted. 

Hemalum is used for staining tissues in bulk. Twenty- 
four hours are required for large pieces. 

Heidenhain's Hematoxylin Stain. — 1. Stain twenty- 
four to forty-eight hours in a simple \ per cent, aqueous 
solution of hematoxylin dissolved by the aid of heat. 

2. Transfer the sections directly to a J per cent, aqueous 
solution of simple chromate of potassium for twenty-four to 
forty-eight hours, changing the solution frequently until no 
more color is given off by the sections. 

3. Wash thoroughly in water. 

4. Alcohol. 

5. Oil. 

6. Xylol balsam. 

Weigert's Iron Hematoxylin. — 
Prepare two solutions : 

A. Hematoxylin, 
Alcohol, 96 per cent, 

B. Liquor ferri sesquichlorati, 
Water, 
Hydrochloric acid, 



I 


gram 


00 


c.c. 


4 


c.c. 


95 


c.c. 


1 


c.c. 



90 PATHOLOGICAL TECHNIQUE. 

For use mix equal parts of A and B. The mixture is deep 
black and is best prepared fresh each time, although it will 
keep and can be used for several days. 

i. Stain sections for several minutes or longer. 

2. Wash in water. 

3. If a counterstain is wanted, place sections for a few sec- 
onds in the following solution : 

Picric acid, saturated aqueous solution, 100 c.c. 

Acid fuchsin, 1 per cent, aqueous solution, 10 c.c. 

4. Wash in water, alcohol, carboxylol, or other clearing 
reagent, balsam. 

Heidenhain's Iron Hematoxylin. — This staining 
method is particularly useful for the demonstration of the 
centrosome, but also stains nuclei and a variety of other 
structures, according to the degree of differentiation. 

1. Fix in corrosive sublimate, Zenker's fluid, or alcohol. 

2. Stain very thin paraffin sections (not over 5 to 6/i 
thick) in a 2.5 per cent, solution of the violet iron alum 
(sulphate of iron and ammonium) for three to twelve hours. 
The sections should be placed vertical in the solution, so 
that no precipitate may fall on them. 

3. Wash off quickly in water. 

4. Stain in a 0.5 per cent, ripened alcoholic solution of 
hematoxylin for twelve to thirty-six hours. 

5. Wash off in water. 

6. Differentiate in the iron-alum solution, controlling the 
results under the microscope. The section should be washed 
off before each examination in a large dish of tap water, 
which immediately stops the decolorization. 

7. Wash in running water for a quarter of an hour. 

8. Alcohol, xylol, xylol balsam. 

A counterstain with Bordeaux R. before, or with rubin S. 
after, the iron stain is sometimes useful. 

Mallory's Chlorid of Iron Hematoxylin. 1 — The 
results which can be obtained by this method are equally 
quick and satisfactory after all of the usual fixing reagents 
except, perhaps, formaldehyde. 

1 Maliory : The Journal of Experimental Medicine, 1900, v., 18. 



HISTOLOGICAL METHODS. 9 1 

Celloidin or paraffin can be employed for embedding. 

1. Stain sections on the slide for three to five minutes in 
a 10 per cent, aqueous solution of ferric chlorid. 

2. Drain and blot the sections ; then pour over them a 
few drops of a freshly prepared I per cent, aqueous solution 
of hematoxylin. If all of the hematoxylin is precipitated by 
the excess of ferric chlorid, pour off the solution and add a 
fresh supply. In three to five minutes the sections will be 
colored a dark bluish-black. 

3. Wash in water. 

4. Decolorize and differentiate in a J^ per cent, aqueous 
solution of ferric chlorid. The sections should be kept con- 
stantly moving in the solution. The differentiation will be 
complete in a few seconds to one or more minutes. 

5. Wash in water. 

6. Dehydrate in alcohol. 

7. Clear in oleum origani cretici. 

8. Xylol colophonium or balsam. 

In the above directions definite strengths have been 
assigned to the solutions, but they may vary greatly with- 
out affecting the result. The important point is to get the 
sections stained deeply, and then to decolorize slowly. The 
differentiation can be stopped at any moment by transferring 
the sections to water. Sometimes it is advisable to examine 
the sections under the microscope to see if enough color has 
been extracted. 

The strength of the hematoxylin solution is unimportant ; 
it is simply necessary to have enough hematoxylin to com- 
bine with all of the iron in and on the section. The sim- 
plest way is to dissolve by the aid of heat a pinch of the 
crystals in a few cubic centimeters of water. A little ex- 
perience will determine about how much is needed. If a 
solution of hematoxylin more than one or two days old is 
used, the color obtained is grayish-blue, and not so bright. 

This method gives a sharp, permanent, dark-blue stain to 
nuclei ; it also stains fibrin of a grayish to dark-blue color ; 
if the decolorization is not carried too far, the contractile ele- 
ments of striated muscle are brought out very sharply. In 



92 PATHOLOGICAL TECHNIQUE. 

Zenker preparations the red blood-corpuscles appear of a 
greenish-gray color. Connective tissue is tinted a pale yellow. 
The nucleus of the amoeba coli stains sharply by this method. 

Carmine Stains. — The ordinary carmine solutions give 
good nuclear stains, but of the finer details in a specimen 
they bring out much less than a direct alum-hematoxylin 
stain. They are much less used now than formerly, except 
as contrast-stains to bacteria and to fibrin in the methods of 
Gram and Weigert, for which purpose lithium carmine wilL 
usually give the best results. 

Alum Carmine; Alum Cochineal (see pages 71, 72). — 1. 
Water. 

2. Stain in either of the above solutions for five to twenty 
minutes. 

3. Wash thoroughly in water. 

4. Alcohol, 95 per cent. 

5. Oleum origani cretici. 

6. Canada balsam. 

Over-staining does not occur. The solutions cannot be , 
recommended for tissues which stain with difficulty. When 
used for staining in bulk, twenty-four to forty-eight hours are 
required. 

Lithium Carmine (see page 72). — 1. Water. 

2. Stain two to five minutes. 

3. Transfer directly to acid alcohol, one or more changes 
for several minutes or more, until the sections are well differ- 
entiated. 

4. Wash in water. 

5. Alcohol, 95 per cent. 

6. Oleum origani cretici. 

7. Canada balsam. 

This method gives an intense and permanent bright-red 
nuclear stain. Over-staining is impossible. A trace of 
picric acid added to the alcohol used for dehydration 
affords a beautiful contrast-stain. 

Acid alcohol, 

Hydrochloric acid, 1 c.c. ; 

70 per cent, alcohol, 99 " 



HISTOLOGICAL METHODS. 93 

Aniline Dyes as Nuclear Stains.— Any of the basic 
aniline dyes may be used as nuclear stains after the following 
general method : 

i. Stain paraffin sections in a strong solution of the dye 
preferred in water or in dilute alcohol for five to thirty 
minutes. 

2. Wash in water. 

3. Dehydrate in absolute alcohol. 

4. Clear in xylol. 

5. Xylol balsam. 

With celloidin sections use 95 per cent, alcohol, blot with 
filter paper, and clear in xylol. 

As a matter of fact, however, certain dyes and certain 
solutions are generally used in preference to the others. 
Most of the colors are more or less affected by all clearing 
reagents except xylol. With paraffin sections and those 
from which the celloidin has been removed it is very 
easy to dehydrate in absolute alcohol and to clear in 
xylol. With celloidin sections, however, this is impossible, 
because the absolute alcohol will dissolve out the celloi- 
din, and this is usually not desirable. For celloidin sec- 
tions, therefore, blot with filter paper, and then pour on 
xylol ; repeat the blotting, followed by xylol, two or three 
times until the specimen is perfectly clear. Mount in xylol 
balsam. 

In washing out the excess of color it is sometimes found 
advantageous to acidulate very slightly either the water or 
the first alcohol with acetic or hydrochloric acid. This pro- 
cess, if not carried too far, tends to make the nuclear stain 
sharper. 

Safranin is one of the very best nuclear-staining aniline 
dyes. Tissues may be hardened in alcohol, corrosive subli- 
mate, Flemming's, Hermann's, or Zenker's fluids. Any 
one of the solutions of safranin given on pages JJ, 78 may 
be used. 

1. Stain paraffin sections two to five minutes to twenty- 
four hours according to the staining solution and fixing re- 
agent used. 



94 PATHOLOGICAL TECHNIQUE. 

2. Wash in water. 

3. Absolute alcohol, several changes, until the section 
appears properly differentiated. 

4. Xylol. 

5. Xylol balsam. 

For celloidin sections dehydrate in 95 per cent, alcohol, 
and clear by the xylol blotting-paper method. To render 
the stain more precise, a few drops of acid alcohol are some- 
times added to the first alcohol. 

Mallory's Bosin and Methylene-blue Stain. — This stain, 
used on paraffin sections of tissues fized in Zenker's fluid, 
can be recommended as the very best general stain yet 
devised. It is a sharp nuclear stain, and, at the same time, 
brings out with a great deal of differentiation all the various 
other structures in the different tissues. It has been in 
constant use for many years as the routine stain for all tissues 
in the pathological laboratories of the Harvard Medical 
School and Boston City Hospital. 

Fix in Zenker's fluid. 

1. Stain paraffin sections in a 5 per cent, aqueous solution 
of eosin for twenty minutes or longer. Sometimes it is ad- 
visable to get a deeper eosin stain by placing the sections in 
the paraffin oven for fifteen to twenty minutes. 

2. Wash in water to get rid of excess of eosin. 

3. Stain in Unna's alkaline methylene-blue solution (see 
page 74), diluted 1-4 or 5 with water, for ten or fifteen 
minutes. 

4. Wash in water. 

5. Differentiate and dehydrate in a dish of 95 per cent 
alcohol, keeping the section in constant motion, so that the 
decolorization shall be uniform. Control the result under 
the microscope. When the pink color has returned to the 
section and the nuclei are still a deep blue, finish the dehy- 
dration quickly with absolute alcohol. 

6. Xylol. 

7. Xylol balsam. 

For celloidin sections use 95 per cent, alcohol, blot, and 
pour on xylol ; repeat the last two steps, until the specimen 
is clear. 



HISTOLOGICAL METHODS 95 

It is important to get a deep stain with eosin, because the 
methylene-blue washes it out to a considerable extent. The 
eosin must be used first, because methylene-blue is readily 
soluble in an aqueous solution of eosin, and therefore is 
quickly extracted if the eosin is used after it, while on the 
other hand eosin is very slightly soluble in an aqueous solu- 
tion of methylene-blue which is precipitated by any excess 
of eosin. 

The success of this staining method has been found by 
Wolbach to depend on the presence of colophonium in the 
alcohol used for differentiation. This is present in alcohol 
obtained from the barrel, but not in alcohol preserved in 
glass. It must, therefore, be added. This is most easily 
done by keeping on hand a 10 per cent, solution of colo- 
phonium in absolute alcohol, and adding a few drops of it to 
the alcohol in which the sections are differentiated. Wolbach 
has also shown that sections fixed in formaldehyde may be 
stained by this method, provided the amount of colophonium 
in the alcohol be increased to from 3 to 10 per cent. 

Diffuse or contrast-stains are useful to make promi- 
nent certain of the tissue-elements left uncolored by the 
nuclear stain. A greater richness of detail is obtained with 
diffuse stains if, after rather deep staining, the sections be 
washed out for some time in alcohol, because certain struc- 
tures possess a greater affinity than others for certain diffuse 
stains, and by holding them are brought out sharply. 

Of the diffuse stains, eosin, picric acid, and acid-fuchsin in 
Van Gieson's mixture are the ones most frequently em- 
ployed. 

Eosin is most frequently used as a contrast to alum-hema- 
toxylin and methylene-blue stains, but is often serviceable 
with alum-cochineal, methyl-violet, etc. It brings out par- 
ticularly well red blood-corpuscles and smooth and striated 
muscle-fibers. The strength of the solutions used after 
hematoxylin varies from ^ to J per cent., according to the 
tissue and the fixative used. Zenker's preparations stain in- 
tensely in eosin, so that for them a very dilute solution is 
advisable. When desired as a contrast-stain to basic aniline 



96 PATHOLOGICAL TECHNIQUE. 

dyes, eosin should be used first in a 5 per cent, solution, be- 
cause otherwise it is likely to be washed out by the nuclear 
stain. 

Picric acid is used for contrast with the carmine stains, 
more rarely with alum-hematoxylin. Striated muscle-fibers 
and cornified epithelium are rendered especially prominent 
by it. To stain with picric acid it is only necessary to add a 
few drops of a saturated aqueous solution to a dish of water, 
or of a saturated alcoholic solution to a little alcohol, and 
allow sections to remain in the solution for a few seconds. 

Van Gieson's stain (see p. 79), a mixture of picric acid 
and acid fuchsin, is excellent as a contrast-stain to alum- 
hematoxylin, especially when it is desirable to render promi- 
nent connective-tissue fibrillae or certain pathological prod- 
ucts. The nuclear stain with alum-hematoxylin must be 
rather deep, because the picric acid to some extent extracts 
or overpowers it. 

1. Stain deeply in alum-hematoxylin. 

2. Wash in water. 

3. Stain in Van Gieson's solution three to five minutes. 

4. Wash in water and dehydrate directly in 

5. Alcohol, 95 per cent. 

6. Oleum origani cretici. 

7. Xylol balsam. 

Neutral Carmine (see page 72). — Neutral carmine is a 
diffuse stain, and is employed more especially for the central 
nervous system and for bone. 

Filter one or two drops of the solution into 20 c.c. of dis- 
tilled water, and leave the sections in the dilute solution over 
night. It is advisable to place a piece of filter-paper on the 
bottom of the dish for the sections to rest on, otherwise they 
may be stained on the upper side only. In double stains 
with hematoxylin and carmine the sections should be stained 
first in the hematoxylin and then thoroughly washed in water 
for six to twelve hours before they are stained in the carmine. 
After the carmine they are again to be thoroughly washed in 
water. 

Combination Stains. — Biondi-Heidenhain Stain (see 
P- 79)- — Tissues must be hardened in corrosive sublimate. 



HISTOLOGICAL METHODS. 9/ 

1. Stain paraffin sections six to twenty-four hours with th? 
dilute solution. 

2. Wash out a little in 90 per cent, alcohol. 

3. Dehydrate in absolute alcohol. 

4. Xylol. 

5. Xylol balsam. 

It is important to place the sections directly from the 
staining fluid into the alcohol, because water washes out the 
methyl-green almost instantly. 

Staining" in Mass. — The staining of tissues in mass is a 
procedure much less employed in pathological than in nor- 
mal histology, but still occasionally useful. For patholog- 
ical tissues a variety of stains is generally necessary. It is 
therefore much better to make a series after one of the 
methods described, and then to stain the sections in what- 
ever way seems best. 

For staining in bulk, only a limited number of solutions 
are available — either those, like alum-carmine and alum- 
cochineal, which do not stain beyond a certain point, or 
those, like lithium and borax-carmine and Heidenhain's 
hematoxylin, which may be decolorized so as to leave only 
the nuclei stained, The process of staining differs from that 
for sections only in the length of time required for each step. 
Tissues \ cm. thick will need from one to two days in the 
staining solution. 



MITOSIS. 

For the study of karyomitosis it is important that the 
tissue be perfectly fresh — that is, just removed from a living 
animal or from one just dead — and that it be fixed in a 
suitable reagent as quickly as possible. The best results 
cannot be obtained with tissues put into a hardening fluid 
over half an hour after removal from a living animal. On 
the other hand, mitotic figures can be demonstrated in tissues 
which have been dead for some time (twenty-four hours or 
more) before being put into a fixing reagent, but the details 
of the figures are not so perfect as those in perfectly fresh 
7 



98 PATHOLOGICAL TECHNIQUE. 

tissues, and the figures are not so numerous, because some 
of them have completed their changes and can no longer be 
recognized. It is therefore evident that mitosis can be 
studied much better in tissues from the lower animals, or 
in tissues removed by operation from the human body, 
than in the organs and tissues removed at post-mortem 
examinations. 

The choice of fixing reagents for the study of mitotic 
figures is important. The figures can often be demonstrated 
after hardening in alcohol or even in Miiller's fluid, but for 
their careful study quicker and more perfect fixing reagents 
must be used. Nearly all of the reagents employed pene- 
trate slowly, so that it is absolutely necessary for the best 
results that the tissue to be hardened be cut into very thin 
slices, rarely over 4 mm. in thickness and preferably not over 
2 mm. The amount of fixing reagent used should always be 
at least ten to fifteen times as great as the volume of the 
tissue, and should be changed if it becomes cloudy. 

The most important fixing reagents are — 

1. Flemming's solution. 

2. Hermann's solution. 

3. Pianese's solution. 

4. Zenker's fluid. 

5. Corrosive sublimate. 

6. Orth's fluid. 

The first three solutions penetrate with much difficulty, so 
that tissues placed in them should be especially thin. The 
most generally useful stain for mitosis is probably safranin. 
The time of staining varies with the solution used. Babes' 
is the quickest. The mitotic figures should be stained 
deeply : then, when treated with alcohol slightly acidulated 
with hydrochloric acid, they will retain the color, while the 
resting nuclei will yield up most of theirs and become very 
pale or even colorless. In consequence of this intense stain 
mitotic figures can then be very readily found. 

Fixation in Zenker's fluid and staining in phosphotungstic- 
acid hematoxylin can be highly recommended. Centrosomes 
and spindles are brought out with great distinctness. 



HISTOLOGICAL METHODS. 99 

Directions for Staining Karyomitotic Figures with 
Safranin. — I. Stain paraffin sections five minutes to twenty- 
four hours, according to solution used. 

2. Wash in water. 

3. Wash in 95 per cent, alcohol to which are added a few 
drops of acid alcohol. 

4. Wash in pure 95 per cent, alcohol, followed by absolute 
alcohol. 

5. Xylol. 

6. Xylol balsam. 

For celloidin sections dehydrate in 95 per cent, alcohol, 
blot, and pour on xylol ; repeat the last two steps until the 
specimen is clear. Safranin can be used after any of the 
above fixing reagents. 

Other useful stains are carbol-fuchsin and aniline-methyl- 
violet, used in the same way as the safranin. The Gram- 
Weigert method gives good results after Flemming's solu- 
tion. 

After fixing in corrosive sublimate mitotic figures can be 
demonstrated by the Biondi-Heidenhain solution, which 
stains resting nuclei blue-violet and mitotic figures green. 
After Pianese's solution his special staining mixtures should 
be used (see page 80). His methods are said to give beau- 
tiful results. 

METALLIC STAINS OR IMPREGNATIONS. 

Experimental investigation has shown that certain metals 
can be used for staining certain tissue-elements, either be- 
cause they are directly reduced from solutions of appropriate 
salts or because they are taken up and retained by certain 
tissue-elements, which are rendered prominent when the 
metallic salt is reduced later. The most valuable metals for 
this purpose are silver, gold, and osmium. 

Silver is used, generally in the form of silver nitrate, to 
stain of a brown or dark-brown color the cement substance 
between epithelial and endothelial cells and the ground sub- 



100 PATHOLOGICAL TECHNIQUE. 

stance of connective tissue. The method finds its chief use 
in pathology in demonstrating the endothelial covering of a 
doubtful surface, in outlining the endothelial cells of patho- 
logically altered blood- and lymph-vessels, in demonstrating 
the treponema pallidum by Levaditi's method, and in staining 
the ground substance of the connective tissue of the cornea 
when that organ is used experimentally for the study of in- 
flammation. In combination with certain other salts, espe- 
cially bichromate of potassium, nitrate of silver is much em- 
ployed in the Golgi methods to stain ganglion-cells and their 
processes in the central nervous system. 

The difficulty of the silver method lies in the fact that the 
salt forms with albuminous fluids granular and thread-like 
coagula which can easily give rise to false pictures. For 
this reason the method is limited almost entirely to natural 
surfaces, which should be washed off with water or a 2 
per cent, solution of nitrate of sodium before the silver solu- 
tion is applied. It is generally advisable to use the nitrate 
of silver in a very dilute solution, I : 250 or 500. The solu- 
tion is allowed to act on the surface for about a minute, and 
is then washed off with water. The tissue is next exposed 
in water to the action either of sunlight or of diffuse light. 
The outlines of the cells soon appear as dark lines, brown to 
black in color. The tissue to be stained should be kept 
stretched, because a precipitation of the silver occurs 
wherever there is a fold in the surface. Although nitrate 
of silver penetrates but a slight distance, it is possible to 
stain the outlines of the endothelial cells of the lymphatics 
and blood-vessels as well as the ground substance of the 
connective tissue — in a rabbit's diaphragm, for instance — by 
treating the upper or lower surface with the silver solution. 
The thoracic organs should be removed, and then the upper 
surface of the tendinous portion of the diaphragm left in 
situ is exposed to the action of the silver salt in the manner 
already described. 

The outlines of the endothelial cells of blood-vessels are 
usually stained by injections of the silver salt through an 
artery. In the same way the limits of the epithelial cells 



HISTOLOGICAL METHODS. IOI 

of the alveoli of the lung can be stained by injections through 
a bronchus. 

Although generally employed in solution, nitrate of silver 
is sometimes used in the solid form, and for the cornea this 
method is preferable. Chloroform the animal, preferably a 
rabbit, deeply; rub the cornea with a stick of nitrate of 
silver hard enough to remove the surface epithelium. Allow 
the salt to act about ten minutes, then kill the animal, re- 
move the eye, cut out the cornea, wash it, and expose to 
diffuse daylight for half an hour. It is then placed in a 
mixture of glycerin and water, 30 parts to 70, very slightly 
acidulated with acetic acid (about y 1 ^ per cent.) for twenty- 
four hours, so as slightly to swell and to soften the tissues. 
Sections of the cornea are best made with the freezing 
microtome. Incise the periphery a little at four points 
equally distant from each other, so that the cornea will lie 
flat. A direct stain with alum-hematoxylin gives by all odds 
the best results. The sections may be mounted in glycerin 
or balsam. The latter method is perhaps the better. De- 
hydrate the sections in 50 per cent., then in 70 per cent., 
alcohol, clear in aniline oil, wash with xylol, and imbed in 
balsam. This method avoids the shrinkage which is caused 
by using strong alcohol. 

Gold, in the form of the simple or double chlorid, is em- 
ployed to stain the cytoplasm of cells of connective tissue, 
and more particularly the axis-cylinders of nerve-fibers and 
their terminal processes. Like nitrate of silver, it acts as a fix- 
ing and hardening reagent as well as a stain. Unfortunately, 
it penetrates tissues but a very slight distance, and, so far as 
staining is concerned, is inconstant in action. Its chief use 
in pathology is in connection with experimental work on the 
cornea and in regeneration. The conditions under which 
the reduction of the gold salt takes place are not exactly 
understood, but both penetration and reduction are aided 
by the action of organic acids, such as formic, citric, and 
tartaric acids, on the tissues both before and after the treat- 
ment with the gold salt. Of the many methods proposed, 
the following are recommended; 



102 PATHOLOGICAL TECHNIQUE. 

Lowit's Formic-acid Method. — i. Place very small bits 
of fresh tissue in a mixture of formic acid I part, and water 
i to 2 parts, until they become transparent (a few seconds 
to several minutes). 

2. Transfer to chlorid of gold, I to 1.5 parts to 100 of 
water, for fifteen minutes. 

3. Formic acid, 1 part to water 3 parts, for twenty-four 
hours. 

4. Concentrated formic acid twenty-four hours. Preserve 
in glycerin or balsam. 

All the steps except the first should be performed in the 
dark. 

Ranvier's Formic-acid Method. — 1. Boil together 8 c.c. 
of a 1 per cent, solution of chlorid of gold and 2 c.c. of 
formic acid. When the solution is cold place very small 
bits of tissue in it for one hour, in the dark. 

2. Wash quickly in water. 

3. Expose to diffuse light in a mixture of formic acid 10 
c.c. and water 40 c.c. Reduction takes place slowly (twenty- 
four to forty-eight hours). 

4. Harden in 70 per cent., then 90 per cent., alcohol in 
the dark. 

Osmic Acid (perosmic acid, osmium tetroxid) is used as 
a fixing reagent and for staining fat and myelin, by which it 
is reduced. As osmic acid is quickly reduced by organic 
substances, care must be taken in making up the solution. 
Remove the label from the sealed tube in which the acid 
comes, and place the tube, after cracking off one end, in a 
glass-stoppered bottle containing enough water to make a 
2 per cent, solution. If desired, the tube can be broken 
after it is in the bottle by violent shaking. It should be 
borne in mind that osmic acid is very-irritating to the bron- 
chial mucous membrane. 

In a I or 2 per cent, solution osmic acid is used to stain fat 
in teased preparations or frozen sections of fresh tissues. In 
Marchi's method it is used to stain fat in tissues which have 
been hardened for some time in Muller's fluid. As a fixing 
reagent it is usually combined with other reagents, as in Flem- 



HISTOLOGICAL METHODS. IO3 

ming's solution, both for its property as a fixative and for 
the purpose of staining any fat present. 

Preparations stained in osmic acid may be kept indefinitely 
in alcohol. When sections are mounted they should be 
cleared in chloroform, and preserved in chloroform balsam 
prepared in the manner described elsewhere. Xylol and 
other clearing reagents cause the stain to fade. 

Clearing Reagents. — The object of clearing reagents 
is to render certain tissue-elements more prominent than 
others. This result may be brought about by dilute acetic 
acid (2-5 : 100), which swells up the ground substance, so 
that nuclei, elastic fibers, fat, myelin, and micro-organisms 
are more distinct, or by alkalies, which destroy the cells and 
ground substance and leave only elastic fibers and bacteria 
but little changed. This method is used almost wholly for 
fresh tissues. 

The same result is more commonly obtained by soaking 
the tissues in substances which by reason of their high index 
of refraction render the tissues more or less transparent. 
Any structure which it is desirable to study is usually pre- 
viously stained and thus easily rendered prominent. This 
second method is most applicable to hardened tissues. 

For soaking and clearing the tissues a variety of reagents 
of different chemical properties are used. Glycerin and 
acetate of potash are not so much employed as formerly, 
because balsam mounts are more generally preferred. Of 
the other reagents (ethereal oils and coal-tar products), the 
the choice depends mainly on two factors — the kind of stain 
which has been employed, and the substance in which the 
sections have been imbedded. Many of the clearing re- 
agents either dissolve celloidin or will not clear it from 95 
per cent, alcohol, and nearly all of them will extract aniline 
colors more or less rapidly. 

Most of the clearing reagents can be used after hema- 
toxylin and carmine stains. For celloidin or paraffin sections 
stained by either of them oleum origani cretici, oil of ber- 
gamot, or the mixture of the oils of cloves and thyme is 
recommended in the order given. 



104 PATHOLOGICAL TECHNIQUE. 

For aniline stains the best clearing reagent is xylol, which, 
however, clears directly only from absolute alcohol. It can 
be used, however, for celloidin or other sections dehydrated 
in 95 per cent, alcohol by a simple method original with 
Welch, and lately brought into notice by Weigert. Blot 
the section on the slide with smooth soft filter-paper, and 
then pour on a few drops of xylol ; repeat the blotting, lol- 
lowed by xylol two or three times, and the section will be 
found to be perfectly clear. 

Oleum Origani Cretici. — Colorless to light brown in 
color ; clears readily from 95 per cent, alcohol without dis- 
solving celloidin ; affects aniline colors slowly. Ordinary 
origanum oil is impure oil of thyme, and should not be 
used. 

Oil of Bergamot. — Light green in color ; clears quickly 
from 95 per cent, alcohol ; does not dissolve celloidin, but 
after repeated use of the same lot of oil it will sometimes 
soften it a little. Affects aniline colors slowly, with the ex- 
ception of eosin, which it extracts very quickly. 

Oil of Cloves. — Straw-colored; clears quickly from 95 per 
cent, alcohol ; dissolves celloidin ; extracts aniline colors, 
especially methylene-blue. 

Oil of Thyme. — Colorless ; clears readily from 95 per 
cent, alcohol ; makes sections brittle ; does not dissolve cel- 
loidin ; affects aniline colors. 

Oil of Lavender. — Clears celloidin sections readily from 
95 per cent, alcohol. 

Oil of Cedar-wood. — Pale straw-color ; clears from 95 per 
cent, alcohol, but, unfortunately, clears celloidin sections very 
slowly ; does not affect aniline colors. 

Aniline {Aniline Oil). — Colorless when perfectly pure and 
fresh, but soon oxidizes and turns brown ; does not dissolve 
celloidin ; clears readily from 70 per cent, alcohol ; will clear 
from water by Weigert's method ; extracts aniline colors 
slowly. 

Xylol. — Colorless ; does not dissolve celloidin ; does not 
affect aniline colors ; clears directly only from absolute alco- 
hol ; but will clear even celloidin sections from 95 per cent. 



HISTOLOGICAL METHODS. 



IO: 



alcohol if they be blotted on the slide, and the xylol be then 
poured over them ; the process of blotting followed by xylol 
must be repeated two or three times. 

Dunham's Mixture of the Oils of Cloves and Thyme. — 
Excellent for sections stained in hematoxylin or carmine. 
Not nearly so expensive as pure origanum or bergamot oil. 

Oil of cloves, I part; 

Oil of thyme, 4 parts. 

Filter if cloudy ; clears celloidin sections readily from 95 per 
cent, alcohol without dissolving the celloidin. 

Weigert's Mixture of Carbolic Acid and Xylol. — 

Carbolic-acid crystals, 1 part ; 

Xylol, 3 parts. 

Recommended for clearing thick sections of the central 
nervous system after carmine and hematoxylin stains only. 
The next mixture is more used now-a-days. 
Weig-ert's Mixture of Aniline and Xylol. — 



Aniline, 
Xylol, 



2 parts ; 
I part. 



Mounting Reagents. — The reagents most generally 
used for permanent mounts are Canada balsam, damar, and 
colophonium. Canada balsam is the most expensive, the 
most difficult to prepare properly (unless the very high-priced 
solid form is employed), and the most highly colored. Damar 
may be obtained practically colorless. Colophonium is the 
cheapest, is but slightly colored, and can be highly recom- 
mended. Canada balsam has the highest index of refraction 
of the three, but the difference between them is slight and of 
no practical importance. 

Neutral Balsam. — The mounting reagents are commonly 
acid in reaction and therefore deleterious to many stains. 
On this account it is advisable always to neutralize the solu- 
tions used. This can easily be done by adding thoroughly 
dried anhydrous carbonate of sodium to the solution kept 
warm in the paraffin oven and shaking it repeatedly. After 



106 PATHOLOGICAL TECHNIQUE. 

the sediment has settled to the bottom of the solution the 
supernatant part can be decanted. 

Canada balsam occurs in commerce as a very thick, tena- 
cious, pale, straw-colored fluid. It should be evaporated 
over a water-bath to drive off all volatile substances, which 
might affect aniline colors, until it becomes solid and brittle 
on cooling. Dissolve it then in xylol, which does not affect 
aniline colors, to a rather thin, syrupy consistency. Two 
pounds of Canada balsam will evaporate to about one 
pound ; add xylol enough to make the mixture up to two 
pounds. In this condition it is often called xylol balsam. 

Canada balsam has a high index of refraction, so that tis- 
sues mounted in it become very transparent, and only those 
parts are visible which are stained. Other solvents of Canada 
balsam, such as chloroform and benzine, may be used, but 
cannot be recommended for sections stained with aniline 
colors. For tissue stained with osmic acid, chloroform bal- 
sam, prepared in the same way as xylol balsam, should 
always be used, otherwise the osmic acid stain will fade 
rapidly. 

Colophonium occurs commercially in the solid form: the 
lightest colored masses should be chosen. Two solutions 
should be prepared, one in xylol for aniline dyes and other 
stains, and one in chloroform for osmium preparations. 
For Wright's blood-stain use a solution of colophonium in 
oil of turpentine (of the best quality). 

Damar also occurs in solid masses, of which the colorless 
pieces should be selected. Dissolve in xylol and then filter. 
If the solution is too thin, evaporate to the proper consist- 
ence. The only fault to be found with damar is that the 
xylol solution sometimes becomes cloudy. The reason for 
this cloudiness is not apparent, but it may be removed by 
filtering again. 

Oil of cedar is recommended as the best mounting re- 
agent after Giemsa stains. The thick evaporated form 
prepared for use with oil-immersion lenses should be em- 
ployed. 



HISTOLOGICAL METHODS. 107 

SPECIAL STAINS FOR CERTAIN TISSUE-ELEMENTS 
OTHER THAN NUCLEI. 

MITOCHONDRIA. 

Mitochondria 1 is a term applied to certain threadlike 
granules, which occur in the cytoplasm of many kinds of 
cells and which have received various names. They may be 
round or oval, rod-shaped, or in the form of filaments. They 
disappear quickly after death and are destroyed by acetic 
acid and most fixing solutions. Special fixatives and stain- 
ing methods are required for their demonstration. The best 
method is that devised by Bensley. 

A. Acetic-osmic bichromate fixation. 

1. Fix very thin sections of perfectly fresh tissue (1 mm. 
thick) for twenty-four hours in the following solution : 

Osmic acid, 2 per cent, solution, 2 c.c. ; 

Potassium bichromate, 2.5 per cent, solution, 8 " 
Glacial acetic acid, 1 drop. 

2. Wash in distilled water for one hour. 

3. Dehydrate in graded alcohols, 50, 70, 95, and 100 per 
cent., twenty-four hours each. 

4. Imbed in paraffin. 

5. Cut sections not over 4 microns thick and attach them 
to the slide by the albumin-water method. 

B. Staining. 

1. Pass sections through toluol or xylol and graded alco- 
hols to water. 

2. One per cent, solution of permanganate of potassium 
for thirty to sixty seconds. 

3. Five per cent, solution of oxalic acid for thirty to sixty 
seconds. 

4. Wash thoroughly in water. 

5. Stain for six minutes at 60 ° C. in Altmann's aniline acid 
fuchsin solution. 

1 Cowdry, E. V., "The Relation of Mitochondria and Other Cytoplasmic 
Constituents in Spinal Ganglion Cells of the Pigeon," Intemat. Monatsschrift 
f. Anat. u. Phys., I-32, xxix., 191 2. 



108 PATHOLOGICAL TECHNIQUE. 

Aniline water, ioo c.c. ; 

Acid fuchsin, 20 grams. 

6. Rinse in distilled water. 

7. Differentiate by dipping for an instant in a 1 per cent, 
aqueous solution of methyl green. 

8. Drain and dehydrate directly and quickly in absolute 
alcohol. 

9. Clear in toluol or xylol and mount in balsam. The 
mitochondria are stained intensely red, the nuclei green. 

MAST=CELLS. 

Mast-cells are found in the tissues under a variety of con- 
ditions, both normal and pathological. They are often num- 
erous in chronic inflammatory processes and occasionally 
occur abundantly in leiomyomata. Their cytoplasmic gran- 
ules stain intensely, like bacteria with the basic aniline dyes, 
especially after fixation in alcohol or formaldehyde. In tissue 
preserved in Zenker's fluid they usually do not stand out 
prominently. In eosin-methylene-blue preparations they 
appear like poorly stained eosinophiles. Several methods of 
staining the granules are given. With Unna's stains for 
plasma-cells a differential color-stain is obtained for the 
granules of the mast-cells. 

Ehrlich's Method. — A. General Stain.—- Harden in alcohol. 

1. Stain with a saturated aqueous solution of dahlia. 

2. Wash out with acidified water. 

3. Dehydrate in 95 per cent, alcohol, absolute alcohol, 
xylol, xylol balsam. 

B. Specific Stain. — Only the cytoplasmic granules are 
stained. Harden in alcohol. 
I. Stain twelve hours in — 

Absolute alcohol, 50 c.c. 

Water, 100 " 

Glacial acetic acid, 12.5 " 

Dahlia, q. s., so that the 
solution is almost saturated. 



HISTOLOGICAL METHODS. 



IO9 



2. Wash out in 95 per cent, alcohol, absolute alcohol, 
xylol, xylol balsam. 

C. Ehrlich-Westphal Method. — Nuclei fed; granules blue. 
Harden at least a week in alcohol. 

1. Stain in the following solution twenty-four hours : 
Alum-carmine solution, 200 ; 
Saturated solution of dahlia in absolute alcohol, 200; 
Glycerin, 100 ; 
Glacial acetic acid, 20. 

(Stir repeatedly, then allow the mixture to stand for some 
time.) 

2. Decolorize for twenty-four hours in absolute alcohol. 

3. Xylol, xylol balsam. 

Unna's Isolated Stains for Mast-cells. — Harden in alco- 
hol. Nuclei blue ; cytoplasmic granules of mast-cells red. 

A. — 1. Stain in polychrome methylene-blue solution, plus 
a little alum, for three hours to over-night. 

2. Wash in water. 

3. Absolute alcohol, xylol, xylol balsam. 

B. — 1. Stain in polychrome methylene-blue solution one- 
quarter of an hour. 

2. Wash in water. 

3. Decolorize in glycerin-ether mixture for five to ten 
minutes. 

4. Wash a long time in water. 

5. Absolute alcohol, xylol, xylol balsam. 



PLASMA=CELLS. 

Plasma-cells arise from lymphocytes. They are often 
abundant in subacute and chronic pathological processes, 
and are characterized by cytoplasm which stains quite deeply 
in alkaline methylene-blue solutions. The eosin-methylene- 
blue stain after fixation in Zenker's fluid brings them out 
very sharply. The two methods best suited for their demon- 
stration furnish at the same time a differential color-stain for 



HO PATHOLOGICAL TECHNIQUE. 

mast-cells. The granules of the latter are stained red, the 
plasma-cells are stained blue. 

Unna's Differential Stains for Plasma-cells and Mast- 
cells. — Harden tissues in absolute alcohol. 

A. — I. Stain paraffin sections in polychrome methylene- 
blue one-quarter of an hour to over-night. 

2. Decolorize in a small dish of water, to which are added 
a few drops of glycerin-ether mixture. 

3. Wash thoroughly in water. 

4. Absolute alcohol, xylol, balsam. 

B. — 1. Stain in polychrome methylene-blue solution five 
to fifteen minutes. 

2. Wash in water. 

3. Decolorize and dehydrate in a \ per cent, alcoholic 
solution of neutral orcein (about fifteen minutes). 

4. Absolute alcohol, xylol, balsam. 
Methyl-green-Pyronin Stain (Unna-Pappenheim). 

Methyl-green, 0.15; 

Pyronin, O.25 ; 

Alcohol, 2.50; 

Glycerine, 20.00 ; 

0.5 per cent, carbol-water to 100.00. 

Fix in alcohol, stain five to ten minutes in incubator, wash 
in cold water, differentiate and dehydrate quickly in absolute 
alcohol, clear in xylol, and mount in xylol balsam. Acetone 
can be used to advantage in place of alcohol to dehydrate, 
because it has less tendency to extract the pyronin from the 
cytoplasm of the cells. 

Schridde's Method for Demonstrating Granules (Mito- 
chondria ?) in the Cytoplasm of Plasma-cells and 
Lymphocytes. 

1. Fixation of thin slices of perfectly fresh tissue from 
operations in Orth's fluid warmed to 35 ° C. and kept at that 
temperature, 24 hrs. 

2. Muller's fluid at room temperature, 24 to 48 " 



HISTOLOGICAL METHODS. 



I I 



3. Running water, 24 hrs. 

4. Distilled water, 6 

5. One per cent, aqueous solution of osmic 

acid in the dark, 24 

6. Running water, 12 

7. Graded alcohols, l inthedark . 

8. Chloroform, J 

9. Imbed in paraffin. 

1. Stain thin sections attached to the slide by albumen 
fixative in Altmann's aniline acid fuchsin solution, 

2 to 24 hrs. 
Acid fuchsin, 20 g. 

Aniline water, 1 00 c.c. 

2. Drain off stain and differentiate in the following solution : 

Sat. ale. sol. of picric acid, 1 part ; 

Twenty per cent, alcohol, 7 parts 

until the section acquires a clear yellowish-red 

color. 

3. Dehydrate in alcohol. 

4. Xylol. 

5. Xylol balsam. 

The neutrophilic granules are stained red; the acidophilic, 
blue. 



THE COLLAGEN FIBRILS AND RETICULUM OF CONNECTIVE 

TISSUE. 

Several methods are available for the demonstration of col- 
lagen fibrils and reticulum. The simplest is by means of 
Van Gieson's picric acid and acid-fuchsin solution, but it is 
applicable to the coarser fibers only. The stain with aniline 
blue is believed to be better than any yet proposed, but is 
limited to tissues hardened in Zenker's fluid. 

A. Mallory's Aniline Blue Stain. — The following method 
is not absolutely differential because, besides collagen fibrils 
and reticulum, it also stains certain hyaline substances, but 



112 PATHOLOGICAL TECHNIQUE. 

these latter are usually so different morphologically that 
confusion cannot arise. The method is also useful for the 
study of fibrin, fibroglia fibrils, smooth and striated muscle- 
fibers, and amyloid. 

i. Fix in Zenker's fluid. 

2. Imbed in celloidin or paraffin. 

3. Stain sections in a 0.5 per cent, aqueous solution of 
acid fuchsin for five minutes or longer, depending on the 
freshness of the tissue. 

4. Transfer directly to the following solution and stain 
from ten to twenty minutes or longer : 

Aniline blue soluble in water (Grubler), 0.5 ; 

Orange G (Grubler), 2.0; 
One per cent, aqueous solution of phos- 

phomolybdic acid, 100.00. 

5. Wash and dehydrate in several changes of 95 per cent, 
alcohol. 

6. Clear in xylol. 

7. Xylol balsam. 

For celloidin sections use 95 per cent, alcohol and clear 
by the xylol blotting-paper method. 

The collagen fibrils and reticulum of connective tissue, 
amyloid, mucus, and certain other hyaline substances stain 
blue ; nuclei, cytoplasm, fibroglia fibrils, axis-cylinders, neu- 
roglia-fibers, and fibrin red ; red blood-corpuscles and myelin- 
sheaths yellow ; elastic fibers pale pink or yellow. The 
various structures do not stain with equal intensity, so that 
certain ones are brought out with great sharpness. This is 
particularly true of the collagen fibrils and reticulum of con- 
nective tissue, and of fibrin and smooth and striated muscle- 
fibers. 

If it is desired to bring out the collagen fibrils as sharply 
as possible, omit the staining with acid-fuchsin. Then the 
nuclei and protoplasm stain yellow, and the blue fibrillae and 
reticulum stand out more prominently. 

B. Van Gieson's Stain. — The proportions given are those 



HISTOLOGICAL METHODS. 1 1 3 

recommended by Freeborn. Occasionally it will be found 
necessary to increase the proportion of the acid fuchsin. 

1. Harden in chrome salts or in corrosive sublimate. The 
results after alcohol are not so good. 

2. Stain deeply in alum-hematoxylin. 

3. Wash in water. 

4. Stain for three to five minutes in 

I per cent, aqueous solution of acid fuchsin, 5 c.c. 
Saturated aqueous solution of picric acid, I OO " 

5. Dehydrate in 95 per cent, alcohol. 

6. Oleum origani cretici. 

7. Xylol balsam. 

C. Unna's Orcein Stain. — 1. Harden in alcohol. 

2. Stain in the concentrated solution of polychrome- 
methylene-blue five minutes. 

3. Wash in water. 

4. Decolorize, differentiate, and stain in a 1 per cent, solu- 
tion of orcein in absolute alcohol fifteen minutes. 

5. Wash in absolute alcohol. 

6. Xylol. 

7. Balsam. 

Nuclei, dark blue ; cytoplasm, pale blue ; elastic and con- 
nective-tissue fibers, deep orcein red; smooth muscle-fibers, 
bluish ; mast-cell granules, red ; cytoplasm of plasma-cells, 
deep blue. 

D. Bielschowsky's Silver Stain. — His method for stain- 
ing collagen fibrils is the same as for axis cylinders, except 
that step 7, the treatment with acetic acid, is omitted. The 
collagen fibrils are stained intensely black on a clear back- 
ground. The method is highly recommended by Herx- 
heimer. 

E. Mall's Differential Method for Reticulum. — 1. Digest 
frozen sections of fresh tissue, 40 to 80 y. thick, for twenty- 
four hours in the following solution : 

Parke, Davis & Co.'s pancreatin, 5 grams ; 

Bicarbonate of sodium, 10 " 

Water, 100 c.c. 



114 PATHOLOGICAL TECHNIQUE, 

2. Wash carefully in clean water. 

3. Place sections in a test-tube half full of water, and 
shake thoroughly in order to remove all the cellular debris. 

4. Spread out on slide, and allow to dry. 

5. Allow a few drops of the following solution to dry on 
surface : 

Picric acid, 10 grams ; 

Absolute alcohol, 33 c.c. ; 

Water, 300 " 

6. Stain for about half an hour in the following solution : 

Acid fuchsin, 10 grams ; 

Absolute alcohol, 33 c.c; 

Water, 66 " 

7. Wash in the picric acid solution for a moment 

8. Alcohol, xylol, balsam. 

FIBROGLIA FIBRILS. 

Connective-tissue cells or fibroblasts are characterized by 
the production of two kinds of fibrils, fibroglia fibrils, which 
bear the same relation to the connective-tissue cells that 
neuroglia fibrils bear to glia cells, and collagen fibrils, which 
are independent of the cells and occur between them. Fibro- 
glia fibrils can be studied to best advantage in actively grow- 
ing connective tissue, for example, in chronic salpingitis and 
in the stroma of carcinomata ; but they are found well devel- 
oped in other situations also, as, for example, in the capsules 
of Pacinian corpuscles. 

Fibroglia fibrils can be stained differentially by several 
different methods. The two simplest and most useful are 
phosphotungstic-acid hematoxylin and the aniline blue stain 
after fixation in Zenker's fluid. The fibrils are often stained 
intensely with eosin, in eosin methylene-blue preparations, if 
the tissue was perfectly fresh when fixed. 

A. Mallory's Phosphotung-stic-acid Hematoxylin Stain. 
— Follow the directions given for neuroglia fibrils on page 
143- 



HISTOLOGICAL METHODS. 115 

B. Mallory's Aniline Blue Stain. — (See directions on 
page in.) 

C. Mallory's Acid-fuchsin Stain. — 1. Fix in Zenker's 
fluid. The tissue should be as fresh as possible, and cut into 
thin sections (2 to 4 mm. thick) for the best results. 

2. Stain celloidin or paraffin sections in a 1 per cent, 
aqueous solution of acid fuchsin overnight in the cold, or 
twenty to thirty minutes in the paraffin oven (56 C). 

3. Wash quickly in water (not over five seconds). Water 
extracts acid fuchsin very rapidly. 

4. Differentiate in a 0.25 per cent, aqueous solution of 
permanganate of potassium for twenty to forty seconds. 
This step must not be prolonged beyond the exact time 
needed or the section will be decolorized. 

5. Wash quickly in water (not over five seconds). 

6. Dehydrate in alcohol. 

7. Clear in xylol. 

8. Mount in xylol balsam. 

While not an absolutely differential stain for these fibrils, 
the method, for the most part at least, is perfectly satisfac- 
tory. It stains intensely red, not only these fibrils and the 
cell nuclei, but also fibrin, the contractile elements of striated 
muscle-cells, the differentially staining fibrils of smooth mus- 
cle-cells, neuroglia fibers, and the cuticular surfaces of epi- 
thelial cells. The collagen fibrils of connective-tissue cells 
appear from brownish-yellow to colorless; elastic fibrils, 
unless degenerated, are bright yellow. 



ELASTIC FIBERS. 

Elastic fibers are not affected by dilute caustic soda or 
potash, or by acids. These reagents are often used, there- 
fore, to demonstrate elastic fibers in the fresh condition, as, 
for example, in sputum, because they render them prominent 
by clearing or destroying the other tissues. The fibers show 
a marked affinity for osmic acid, staining with greater rapid- 
ity than most other tissue-elements. 



Il6 PATHOLOGICAL TECHNIQUE. 

For bringing out elastic fibers in sections of hardened 
tissues there are three excellent differential stains. The great 
advantage of Verhoeff's is that it is applicable after Zenker 
fixation. 

A. Weigert's Stain for Elastic Fibers. — Fixation in 
alcohol or formaldehyde is preferable, but other fixing re- 
agents give good results. Imbed in celloidin or paraffin. 
After fixation in Zenker's fluid, sections stain slowly, and 
there is a greater tendency, perhaps, to diffuse coloring of 
the collagen fibrils. 

1. Stain sections twenty minutes to one hour in the fol- 
lowing solution : 

Fuchsin, 2 ; 

Resorcin, 4 ; 

Water, 200. 

Boil the solution in a porcelain dish ; when briskly boil- 
ing add 25 c.c. of liquor ferri sesquichlorati ; stir and boil 
for two to five minutes. A precipitate forms. Cool and 
filter. The filtrate is thrown away. The precipitate remains 
on the filter-paper until all the water has drained away or 
until the precipitate has thoroughly dried. Then return 
filter and precipitate to the porcelain dish, which should be 
dry, but which should contain whatever part of the precipi- 
tate remained sticking to it. Add 200 c.c. of 95 per cent, 
alcohol, and boil. Stir constantly, and fish out the filter-paper 
as the precipitate is dissolved off. Cool ; filter ; add alcohol 
to make up the 200 c.c. Add 4 c.c. of hydrochloric acid. 

2. Wash off in alcohol. 

3. Blot with filter-paper, and add xylol quickly ; repeat 
the blotting, followed by xylol, two or three times until the 
section is perfectly cleared. 

4. Xylol balsam. 

Sections can be stained for several hours. If the rest of 
the tissue is overstained, differentiate in acid alcohol ; if the 
sections are too deeply stained, the color cannot be washed 
out. Diffuse staining can be avoided by diluting the stain 



HISTOL OGICAL ME THODS. 



117 



either with alcohol or, better still, with alcohol containing 
2 per cent, of hydrochloric acid. The elastic fibers appear 
dark blue, almost black, on a clear background. The nuclei 
can be stained red with carmine before or after the staining 
of the fibers. After Zenker fixation, carmine stains are diffi- 
cult to obtain. A light nuclear stain with alum-hematoxylin, 
after the fibrils have been colored, is preferable. 

The solution keeps for months. 

If it be desired to keep sections for some time before 
mounting, wash them in alcohol and place in water. 

B. Hart's Modification of Weigert's Elastic Tissue 
Stain. — 

1. Stain sections in lithium carmine thirty minutes. 

2. Then direct into 

Acid alcohol, 100 c.c. ; 

Weigert's stain, 5 " 

Stain over night; twelve hours at least. 



3. Wash in 85 per cent, alcohol; then dehydrate, clear, and 
mount as in Weigert's method. 

C. Unna's Orcein Method for Elastic Fibers. — Unna's 
latest method of using orcein is as follows, and can be highly 
recommended : 

1. Stain sections in the following solution : 



Orcein (Griibler), 
Hydrochloric acid, 
Absolute alcohol, 



1; 

1; 

100. 



Place the sections in a dish and pour over them enough 
of the solution to cover them. W T arm gently in an incu- 
bator or over a small flame for ten to fifteen minutes until 
the solution thickens, or leave in the solution at room- 
temperature overnight. 

2. Wash off thoroughly in dilute alcohol (70 per cent). 

3. Wash in water to get rid of all the acid and to fix the 
color. 

4. Alcohol. 



Il8 PATHOLOGICAL TECHNIQUE. 

5. Oil. 

6. Balsam. 

The washing in water is not absolutely essential. 

Elastic fibers are stained of a deep silky-brown color, con- 
nective tissue a pale brown. If it is desirable to have only 
the elastic fibers stained, wash for a few seconds in 1 per 
cent, hydrochloric-acid alcohol before washing in water. 
The nuclei can be brought out by staining in Unna's poly- 
chrome methylene-blue solution after washing the sections 
in water. 

Verhoeff's Elastic Tissue Stain. — Fixation in formaline 
or Zenker's fluid preferred. Tissues or sections should not 
be treated with iodine solution before staining. Mercurial 
precipitates, if removable, are removed by the staining solu- 
tion. For the best results the solution should be used within 
twenty-four hours, but satisfactory specimens may be ob- 
tained with solutions one month old. 

The staining fluid is made as follows : 

Hematoxylin crystals, 1 gm. 

Absolute alcohol, 20 c.c. 

Dissolve in test-tube by aid of heat, filter, and add in order 
given : 

Aqueous solution (10 per cent.) of ferric 

chlorid, 8 c.c. 

Lugol's solution (iodin, 2 ; potassium 

iodid, 4; water, 100), 8 c.c. 

Sections are immersed in the staining fluid for fifteen min- 
utes or longer until perfectly black, and are then differen- 
tiated in a 2 per cent, aqueous solution of ferric chlorid. The 
differentiation requires only a few seconds. To observe the 
stages in the differentiation, the sections may be examined 
in water under a low magnification. If the differentiation has 
been carried too far, the sections may be restrained, provided 
that they have not been treated with alcohol. 

The sections are now washed in water, followed by 95 per 
cent, alcohol to remove the stain of the Lugol solution, and 



HISTOLOGICAL METHODS. 119 

then are allowed to remain in water five minutes or longer. 
They are then counterstained in a J per cent, aqueous solu- 
tion of eosin if desired, passed through alcohol, oil of origa- 
num, and mounted in balsam. 

By this method elastic tissue is stained black, while con- 
nective tissue, fibroglia, myoglia, and neuroglia fibrils, myelin, 
and fibrin take the eosin stain. Nuclear staining may be 
obviated by doubling the amount of Lugol's solution in the 
staining fluid. Degenerated elastic tissue (elacin) is also 
stained by this method. The degenerated fibrils may be dis- 
tinguished from the normal by staining less intensely and 
presenting less distinct outlines. 

Equally good results, especially after Zenker's fixation, 
may be obtained by staining the tissues en masse. Myelin, 
however, is also stained. Thin slices of tissue after fixation 
are removed from 80 per cent, alcohol and immersed in the 
staining fluid four days. They are then quickly rinsed in 
water to remove excess of stain, placed in 80 per cent, alco- 
hol, and imbedded in the usual manner. The sections are 
differentiated in a J per cent, solution of ferric chlorid. 

SMOOTH AND STRIATED MUSCLE=CELLS. 

Smooth and Striated Muscle-cells. — For the demon- 
stration of muscle-cells double stains, such as alum-hema- 
toxylin and eosin or eosin and methylene-blue, are sufficient. 

For bringing out the finer details in the cytoplasm, how- 
ever, phosphotungstic-acid hematoxylin and the aniline blue 
stain are much to be preferred. It is imperative that the 
tissue be perfectly fresh, especially if the myoglia fibrils in 
smooth muscle-cells are to be studied, because they very 
quickly undergo postmortem changes. Thin sections of the 
tissues to be studied should be put into Zenker's fluid within 
five to ten minutes at the most after removal from the body, 
if the best results are desired. Autopsy material is practi- 
cally useless. The most desirable tissues are those obtained 
directly at operations on the human body. 

A. Phosphotungstic-acid Hematoxylin Stain (Mallory). 
For directions see page 143. 



120 PATHOLOGICAL TECHNIQUE. 

B. Aniline Blue Stain (Mallory). (See page in.) 

C. Benda's Stain for Myoglia Fibrils.— i. Fix fresh 
material in Zenker's fluid for twenty-four hours. 

2. Wash for a number of hours in water. 

3. Make frozen sections. 

4. Place sections in a 0.5 per cent, solution of chromic 
acid for twenty-four hours. 

5. Wash off in water. 

6. Place in a 0.25 per cent, solution of permanganate of 
potassium for about three minutes. 

7. Wash off in water. 

8. Place in Pal's mixture of sulphite of sodium and oxalic 
acid for five minutes. 

9. Wash off in water; take up section on slide. 

10. Cover with the following solution : 

Crystal-violet, saturated solution in Jo per 

cent, alcohol, 1 part ; 

Acid alcohol, 1 " 

Aniline-water, 2 parts. 

11. Blot with filter-paper. 

12. Cover with dilute Lugol's solution. 

13. Blot with filter-paper ; dry. 

14. Differentiate in aniline oil and xylol, equal parts. 

15. Xylol; xylol balsam. 

THE CENTRAL NERVOUS SYSTEM. 

In the preservation of the central nervous system the 
special structures which require consideration are the gan- 
glion-cells, including both the dendritic and the axis-cylinder 
processes, the myelin-sheaths, and the neuroglia-fibers. No 
one fixing reagent is suited for the best preservation of all 
of them, unless possibly it be formaldehyde. 

The main fixing fluids for the nervous system until within 
a very short time have been various solutions of the chrome 
salts, particularly of bichromate of potassium, either alone 
or in combination with sulphate of sodium, as in the well- 



HISTOLOGICAL METHODS. 121 

known Muller's fluid. The chief objections to the chrome 
salts as fixatives are that they penetrate and harden very 
slowly, and do not preserve properly either the ganglion- 
cells or the neuroglia-fibers. On the other hand, they prob- 
ably preserve the axis-cylinders as well as any reagent we 
yet know, and are invaluable for their property of enter- 
ing into some chemical combination with myelin, in con- 
sequence of which it is possible to obtain by the method 
originated by Weigert a differential stain of the myelin- 
sheaths. 

The new fixing reagent, formaldehyde, seems likely to find 
its greatest use histologically as a fixative of the central 
nervous system. It penetrates and hardens up to a certain 
degree with great rapidity. It also preserves in certain struc- 
tures the special chemical properties on which certain differ- 
ential stains depend. Small pieces of nervous tissue are 
properly fixed in the standard solution (4 per cent, solution 
of formaldehyde gas) in four days. A whole brain will be 
so hardened in ten days to two weeks that thin serial sections 
can be made through it without fear of the slices altering 
their shape in the least. The process could undoubtedly 
be hastened by injecting the arteries. 

It must be borne in mind, however, that for most purposes 
formaldehyde must be followed by other reagents before the 
structures and their chemical properties preserved by it are 
properly fixed so that they will not be altered when trans- 
ferred to alcohol. In other words, formaldehyde may be 
looked upon as a very quick preliminary fixing reagent. 
The hardening of brains entire in it is not recommended, 
except in certain cases — for instance, of cysts, hemorrhages, 
or occasionally of tumors — where the gross lesions and the 
tracts or structures affected by them are of more importance 
than the finer histological changes. For the proper preser- 
vation of ganglion-cells and of neuroglia-fibers very small 
pieces must be taken and fixed by the special methods 
given ; but if the main object is to trace system-degenera- 
tions, much larger pieces, or even the whole brain, may be 
taken, because the myelin-sheaths change comparatively very 
slowly after death. 



122 PATHOLOGICAL TECHNIQUE. 

The stains for the central nervous system may be divided 
into two classes — general and differential. For nearly all of 
them preliminary fixation in formaldehyde is advisable or 
possible. This renders the immediate preservation of ner- 
vous tissue very simple, and at the same time allows a variety 
of mordanting and staining methods to be used later. 

The staining of the various histological elements of the 
central nervous system and the fixing reagents best suited 
for each of them will be considered under the following 
headings : 

General Stains. 

{Cytoplasmic granules ; 
Dendritic and axis-cylinder processes ; 
Axis-cylinders and their terminal processes. 
Stains for the myelin-sheath. 
Stains for the neuroglia-fibers. 

General Stains. — General stains include the ordinary 
nuclear stains, with or without a contrast-stain, and certain 
diffuse single or combined stains which color the nuclei, the 
cell-protoplasm, including to a varying extent the dendritic 
processes of the ganglion-cells, the axis-cylinders, and the 
neuroglia-fibers. The different stains vary somewhat in regard 
to the structures which they bring out most prominently. 

The best fixation for the general stains is Zenker's fluid, to 
be followed by the eosin-methylene blue and the phospho- 
tungstic acid hematoxylin stains. Alum-hematoxylin, fol- 
lowed by eosin, is sometimes useful. The eosin, if deep 
enough, brings out fairly well both the dendrites and the 
axis-cylinders. 

The various carmine solutions, particularly neutral, am- 
monia, and picro-carmine, have long been the favorite diffuse 
stains for the central nervous system, but the uncertainty of 
their action and the difficulty of always getting a good stain- 
ing solution have gradually led to the introduction of more 
reliable methods. Of these, the simplest, quickest, and in 
many ways the most generally useful is — 

A. Van Gieson's Stain.— It may be used after any fixa- 
tion. Although this mixture of acid fuchsin and picric acid 



HISTOLOGICAL METHODS. 123 

may be made up in the way originally recommended, the 
following exact proportions, given by Freeborn for staining 
nervous tissues, will be found generally preferable : 

1 per cent, aqueous solution of acid fuchsin, 1 5 c.c. ; 
Saturated aqueous solution of picric acid, 50 " 
Water, 50 " 

1. Stain sections first rather deeply in alum-hematoxylin. 

2. Wash in water. 

3. Stain in above solution three to five minutes. 

4. Dehydrate in alcohol. 

5. Oil, xylol balsam. 

The nuclei appear bluish red, the ganglion-cells and pro- 
cesses red, the axis-cylinders brownish-red, the myelin- 
sheaths yellow, the neuroglia-fibers orange red, connective- 
tissue fibrillae deep red. After certain reagents this solution 
will not give a sufficiently intense stain. In such cases a 
mixture of 1 part of a 1 per cent, solution of acid fuchsin to 
2 parts of a saturated solution of picric acid is recommended. 

B. Phosphotung-stic-acid hematoxylin (see page 71) 
will be found of much value as a general stain for the central 
nervous system if employed in the manner recommended 
for neuroglia-fibers after fixation by the method there given, 
because a greater differentiation of the various tissue-ele- 
ments is obtained than by any other method. 

C. Phosphomolyb die- acid Hematoxylin (see page 70). 
— This solution stains well only after fixation in a simple 
chrome salt, as in Miiller's fluid. 

1. Stain sections twenty minutes to one hour. 

2. Wash out in two or three changes of 50 per cent, 
alcohol until the celloidin becomes completely decolorized 
(about five minutes). 

3. Dehydrate in 95 per cent, alcohol. 

4. Oil, xylol balsam. 

The ganglion-cells are often overstained, especially if the 
tissue has been hardened but recently. The method is par- 
ticularly good for bringing out the axis-cylinders and the 
neuroglia-fibers. 



124 PATHOLOGICAL TECHNIQUE. 

D. Aniline Blue Stain (see page 73). — The method 
recommended for connective-tissue fibers will also be found 
very useful for the study of the nervous system. The best 
results are obtained after fixation in Zenker's fluid. 

E. Nigrosin. — 1. Stain sections in a concentrated aqueous 
solution of nigrosin five to ten minutes. 

2. Decolorize and dehydrate in weak, then in strong, 
alcohol. 

3. Oil, Canada balsam. 

The stain is not very sharp, but is simple and useful, par- 
ticularly for low-power observation. 

Stains for Nissl or Tigroid Bodies. — These bodies 
are brought out with great sharpness by the eosin-methy- 
lene blue stain after fixation in Zenker's fluid, but the follow- 
ing are the classical methods for demonstrating them : A. 
Nissl's Stain. — 1. Carefully harden pieces of tissue not over 
I to 1.2 cm. in diameter in 96 per cent, alcohol. 

2. Cut sections without imbedding, as follows : Remove 
excess of alcohol from tissue with filter-paper ; dip base of 
specimen in thick celloidin ; mount on block ; harden in 96 
per cent, alcohol. Moisten knife with 96 per cent, alcohol. 
Sections should always be under -^ mm. in thickness. In 
order to be able to compare the number of cells, etc., in one 
case with those in another, the sections should be of uni- 
form thickness. Preserve sections in 96 per cent, alcohol. 

3. Stain the sections in the following solution heated over 
a flame until it bubbles noisily (6o°-yo° C.) : 

Methylene-blue, B patent, 1 3.75 ; 

Venetian soap, l -7S\ 

Distilled water, 1000. 

4. Wash out in — 

Aniline oil, 10 parts ; 

96 per cent, alcohol, 90 

until the color is no longer given off in coarse clouds. 

1 Nissl personally prefers and uses the make of Carl Buchner und Sohn, 
Munich. 



HISTOL O GICAL ME THODS. 



125 



5. Transfer section to slide; dry with filter-paper and 
cover with oil of cajuput. 

6. Blot with filter-paper, and then wash with a few drops 
of benzine. 

7. Add a few drops of benzine-colophonium (made by 
dissolving colophonium in benzine for twenty-four hours and 
then decanting). 

8. Hold the slide above the flame until all the benzine is 
driven off. (Nissl no longer recommends burning off the 
benzine.) 

9. Cover-slip. Warm the slide, so that the colophonium 
will spread out evenly between the cover-slip and the slide. 

The specimen is now mounted in a medium in which dif- 
fusion of color cannot take place, so that the stain is practi- 
cally permanent. The best results are obtained with tissues 
which have not been hardened in alcohol over one to four 
days. Contact with water, weak alcohol, and ether must be 
avoided. 

B. Lenhossek's Stain. — The following method will be 
found simpler, but the specimens are not permanent. 

1. Harden sections in 90 per cent, alcohol, then in 96 per 
cent, or in formaldehyde followed by alcohol. Do not keep 
the tissues too long in alcohol. 

2. Imbed sections in celloidin or paraffin, or cut without 
imbedding, as in Nissl's method. 

3. Stain sections in a completely saturated solution of 
thionin for five minutes. 

4. Wash for a few seconds in water. 

5. Differentiate in aniline, 
Absolute alcohol, 

Do not decolorize too long. 

6. Clear in oleum cajuputi. 

7. Xylol. 

8. Xylol balsam. 

The granulations can be shown by other stains, such as 
safranin, fuchsin, dahlia, alum-hematoxylin. 

Ganglion-cells ; Dendritic and Axis-cylinder Pro- 
cesses. — Golgi's Methods. — Golgi's methods, although of 



I part ; 
9 parts. 



I2 6 PATHOLOGICAL TECHNIQUE. 

the greatest value in the study of the normal histology of 
the central nervous system, are of very little use in the study 
of its pathology. The reason of this is the very peculiarity 
that makes the method of value in normal histology — 
namely, that it picks out here and there a cell and stains it 
with all its wealth of processes more or less completely, 
while the neighboring cells are left colorless. If all of the 
cells and their processes were stained, the picture presented 
would be a confused mass. In pathological histology, where 
the presence or absence of certain cells or processes is of 
paramount importance, it is of primary necessity that every 
cell within a given area shall be perfectly stained. 

Golgi introduced three different methods of obtaining the 
stain now called after his name. They are spoken of as the 
slow, the mixed, and the short methods. Golgi himself 
employed principally the first two methods, and they are 
still used for the study of the developed brain and cord. 

The quick method exclusively has been used by Ramon y 
Cajal and other recent investigators for the study of em- 
bryonic nervous tissue. 

The following points are to be borne in mind : The tissue 
should be as fresh as possible, and should be cut into small 
pieces, not over I to ij cm. thick — for the quick method 
even thinner. With the corrosive-sublimate method, how- 
ever, larger pieces can be used. 

Large quantities of the solutions should be used — at least 
ten times the volume of the specimen It is best to keep 
the specimens in the solution in the dark, especially in using 
the corrosive-sublimate method. 

Golgi' s Slow Method. — i. Harden the tissues in a 2 per 
cent, solution of bichromate of potassium two to six weeks. 
In summer fifteen to twenty days are sufficient ; in winter, 
unless the temperature is kept at 25 ° C, one to one and a 
half months will be required. Keep the specimens in the 
dark. Large amounts of the solution should be used, and 
it should be frequently changed, especially during the first 
week. 

2. Transfer either to (a) a f per cent, solution of nitrate 



HISTOLOGICAL METHODS. 1 27 

of silver for twenty-four to forty-eight hours ; a longer time 
will do no harm ; or to (b) a J per cent, solution of corrosive 
sublimate — small pieces eight to ten days, large pieces two 
months or more. Change the solution frequently during the 
first few days ; later only when the solution gets yellow. 

This second procedure is recommended for larger pieces 
of tissue than can properly be impregnated by (a). 

Golgl's Mixed Method. — 1. Harden small pieces of tissue 
for three to five days or longer in a 2 per cent, solution of 
bichromate of potassium at 25 ° C, in the dark. 

2. Transfix to a mixture of — 

1 per cent, solution of osmic acid, 2 parts ; 

2 per cent, solution of bichromate of potas- 

sium, 8 " 

for three to eight days. 

3. Place in a f per cent, solution of nitrate of silver for 
twenty-four to forty-eight hours. 

Golgl's Quick Method. — 1. Small pieces of fresh tissue 
are placed directly in the following solution : 

1 per cent, solution of osmic acid, 1 part ; 

3.5 per cent, solution of bichromate of potassium, 4 parts, 
for several days (three to eight). 

2. They are then transferred to a large amount of a f per 
cent, solution of nitrate of silver for one, two, or six days. 

The length of time the tissues should remain in the 
osmic-acid and bichromate-of-potassium solution depends on 
what elements it is desired to impregnate. In the human 
cord the time is in general the following: 

1. Neuroglia, 2-3 days ; 

2. Nerve-cells, 3-5 " 

3. Nerve-fibers and collaterals, 5-7 " 

The further treatment of the tissues impregnated by these 
methods is as follows : Alcohol must be avoided as much as 
possible. The tissues are usually firm enough to cut after 
the impregnation ; if not, place in absolute alcohol for fifteen 
to thirty minutes. The sections should be rather thick, ^V 



128 PATHOLOGICAL TECHNIQUE. 

to i 1 ^ mm. They may be made free hand with a razor or in 
the microtome. For either method the tissues can be held 
between pieces of elder-pith, or may be quickly imbedded in 
celloidin by dehydrating for a few minutes in absolute alco- 
hol and then placing in a thick solution for five minutes. 
From the celloidin they are mounted in elder-pith or on 
blocks, and placed for a short time in 80 per cent, alcohol to 
harden. 

Treatment of Sections. — 1. Dehydrate quickly in alcohol. 

2. Clear in oil of cloves or bergamot. 

3. Wash off with xylol. 

4. Mount without a cover-glass in xylol damar, and dry 
quickly at 40 C. 

The mounted sections must be protected from the light 
and from dust as much as possible. Cajal has modified 
Golgi's quick method by repeating the steps (Cajal's so- 
called double method) so as to get a more perfect impregna- 
tion. The same osmic-acid and bichromate-of-potassium 
solution may be used over again, or a fresh solution, con- 
taining about one-half as much osmic acid, is made up fresh. 
The silver solution should be taken fresh each time. Len- 
hossek, Weigert, and others have obtained very good Golgi 
preparations with tissues first fixed in formaldehyde. 

Of the various methods proposed for fixing the Golgi 
stains so that contrast-stains could be used with them and 
the specimens protected by cover-slips, the simplest and 
most practical seems to be that advocated by Kallius. 

The Method of Kallius for Fixing Golgi Stains. — The 
method depends on the employment of a photographic 
developer to reduce the bichromate of silver to metallic 
silver. 

1. Place sections for several minutes in a solution com- 
posed of 1 part of the following developer: 



Hydrochinon, 


1; 


Sulphite of sodium, 


8; 


Carbonate of potassium, 


1.5; 


Water, 


575, 



HISTOLOGICAL METHODS. 



120. 



plus one-third to one-half as much absolute alcohol until 
the sections become gray to black in color. If too much 
alcohol is added, the carbonate of potassium will be pre- 
cipitated, but will redissolve on the addition of a little more 
developer. 

2. 70 per cent, alcohol for ten to fifteen minutes. 

3. Hyposulphite of sodium (20 per cent, aqueous solu- 
tion). 

4. Wash thoroughly in a large amount of water for twenty- 
four hours. 

5. Alcohol, oil, xylol balsam ; cover- glass. 

Cox's Modification of Golgi' s Corrosive-sublimate 
Method. — The same black pictures are obtained by this 
method as by Golgi's, but with this difference, that nearly all 
of the cells in the section are impregnated. This is an ad- 
vantage when the topographical arrangement of the cell- 
layers is desired, but a disadvantage when it comes to the 
study of individual cells, because on account of the luxuri- 
ance of the impregnation such a study is rendered impos- 
sible. Small pieces of nervous tissue are placed in the fol- 
lowing solution : 

Bichromate-of-potassium 5 per cent, solution, 20 ; 

Corrosive-sublimate 5 per cent, solution, 20 ; 

Distilled water, 30-40; 

Simple chromate-of-potassium 5 per cent, solution, 16. 

The time required for impregnation is a month in summer 
and two to three months in winter. The after-treatment is 
the same as for Golgi preparations. 

Axis-cylinders and their Terminal Processes. — 

The three methods most in use in the past for the study of 
central and peripheral nerve-fibers and their terminations are 
the gold, the Golgi, and the methylene-blue methods. All 
three may give beautiful results, but, as a rule, they are very 
unreliable. Their use is confined almost wholly to the study 
of normal tissues. More recently, Bielschowsky's silver im- 
pregnation method has come to the front and has been found 
of considerable value in pathological work. 



130 PATHOLOGICAL TECHNIQUE. 

1. Gold Stain for Nerve-fibers. — For the application of 
the gold method to fresh tissues see p. 101. 

Various attempts have been made to devise a reliable 
method of employing chlorid of gold for staining nerve- 
fibers in sections of hardened tissues. The results have not 
been altogether successful. The best results can probably 
be obtained by — 

A. GerlacJis Method. — 1. Harden tissues in a 1-2 per cent, 
solution of bichromate of ammonium for one to three weeks ; 
cut sections without passing through alcohol, which must be 
avoided. 

2. Place the sections in a very dilute solution (yj^ per 
cent.) of the double chlorid of gold and potassium very 
slightly acidulated with hydrochloric acid, for ten to twelve 
hours, until they become slightly violet in color. 

3. Wash in a solution of hydrochloric acid 1, to water 
2000-3000. 

4. Place for ten minutes in a ^ per cent, solution of hy- 
drochloric acid in 60 per cent, alcohol. 

5. Absolute alcohol, oil of cloves, Canada balsam. 
Another method frequently recommended is the following : 

B. Freud's Gold Stain for Nerve-fibers. — 1. Harden tissues 
in Erlicki's or Miiller's fluid, followed by alcohol. Imbed in 
celloidin. 

2. Stain sections three to five hours in I per cent, solution 
of chlorid of gold, and 95 per cent, alcohol, equal parts. 

3. Wash in water. 

4. Reduce in — 

Caustic soda, 1 ; 

Distilled water, 6, 

for two to three minutes. 

5. Wash in water. 

6. Place for five to fifteen minutes in a 10 per cent, solu- 
tion of iodid of potassium. 

7. Wash in water. 

8. Alcohol, oil, xylol balsam. 



HISTOLOGICAL METHODS. 



131 



2. Methylene-blue Stain for Nerve-fibers. — The methyl- 
ene-blue method is due to Ehrlich. Many modifications of 
the original procedure have been suggested with a view to 
making the results surer or the specimens more permanent. 
Tissues can be stained either by injection or by immersion. 

The methylene-blue used should be Grubler's " rectified 
methylene-blue for vital injection." 

For injection in the blood- or lymph-vessels of live or 
dead animals a 1 to 4 per cent, solution in normal salt solu- 
tion is recommended. The injected organs are exposed to 
the air until a bluish tint is visible. As soon as the greatest 
intensity of stain is reached (five minutes to two hours) the 
color in the preparation is fixed by placing small bits of the 
tissue in a freshly-filtered, cold, saturated, aqueous solution 
of picrate of ammonium, or, better still, in the solution given 
below, recommended by Bethe. 

Very small or thin pieces of tissue intended for staining by 
immersion (the method employed for human tissues) are 
placed in a very dilute solution (^g— IT P er cen t.) of methyl- 
ene-blue in normal salt solution. Lavdowski recommends 
very highly a solution of methylene-blue in egg-albumin, 
either alone or combined with chlorid of sodium or ammo- 
nium. The white of egg is freed from the thicker portions or 
filtered. When the experiment is to last some time, add to 
the egg-albumin an equal part of a J per cent, solution of 
chlorid of sodium or of a \ per cent, solution of chlorid of 
ammonium. The tissue, protected by a large dish, is exposed 
to the air for fifteen minutes to twelve hours, until the maxi- 
mum stain is obtained. 

The stain may then be fixed by the method already given, 
or, better still, in the following manner : 

Bethe's Method of Fixing Methylene-blue Stains of Nerve- 
fibers. — 1. Wash off excess of color with normal salt solution. 

2. Place in — 



Molybdate of ammonium, 
Distilled water, 
Peroxid of hydrogen, 
Hydrochloric acid, 



1 gr.; 
10 c.c. ; 

T " 

I drop. 



132 PATHOLOGICAL TECHNIQUE. 

A precipitate forms on making up the solution, but disap- 
pears on shaking. The solution will keep eight days, but is 
best made up fresh each time. It should be used as cold as 
possible, preferably surrounded by a mixture of ice and salt. 
Leave the tissue in the cold solution for from two to five 
hours, and then for a while longer at the room-temperature. 

3. Wash one half to two hours in running water. 

4. Dehydrate and harden as quickly as possible (not over 
twelve to twenty-four hours) in cold absolute alcohol. (The 
color is soluble in warm alcohol.) 

5. Clear in xylol. 

6. Imbed in paraffin. 

The sections may be mounted directly or brought into 
water and stained with alum-cochineal for contrast. If a little 
osmic acid be added to the fixing solution after the speci- 
mens have been in it for a while, a more permanent methyl- 
ene-blue stain is obtained. 

3. Bielschowsky 's method of staining axis- cylinders and 
neurofibrils by silver impregnation : 

1. Fix in 4 per cent, formaldehyde (10 per cent, formol) 
for twenty-four hours or longer. 

2. Cut very thin sections (5 p) with the freezing micro- 
tome and place them in distilled water. (Bielschowsky has 
recently advised transferring the sections from the water to 
pure pyridin for twenty-four to forty-eight hours and then 
washing thoroughly in distilled water before proceeding with 
step 3). > 

3. Transfer them to a 2 to 3 per cent, solution of nitrate 
of silver in distilled water for twenty-four, preferably forty- 
eight, hours. 

4. Wash quickly in distilled water. 

5. Place the sections in the following solution for two to 
three minutes (as a rule, about five minutes) : 

To 10 c.c. of a 10 per cent, aqueous solution of nitrate of 
silver add 5 drops of a pure 40 per cent, aqueous solution of 
sodium hydrate. A precipitate is formed, which is to be dis- 
solved by adding ammonia drop by drop while stirring con- 
stantly with a glass rod. The ammonia must not be added 
in excess, but only enough to dissolve the precipitate. It is 



HISTOLOGICAL METHODS. 133 

best to leave a trace of precipitate and filter. Add to the 
filtrate enough distilled water to make the solution measure 
20 c.c. This solution contains the easily reducible silver salts, 
ammonium nitrate of silver, and ammonium oxid of silver. 

6. Wash in distilled water. 

7. Place in weak acetic acid (5 drops of glacial acetic acid 
to 20 c.c. of distilled water) until the color of the sections 
changes from brown to dark yellow. 

8. Transfer the sections to an 8 per cent, solution of for- 
maldehyde (20 per cent, formol) for five to thirty minutes 
until no more white clouds are given off. In this solution 
reduction to metallic silver occurs. The sections appear 
brownish to black, depending on how long they were washed 
in the distilled water. 

The sections are now toned with gold in order to intensify 
the stain of the fibrils and to render it more permanent. 

9. Wash quickly in distilled water. 

10. Place sections in the following gold bath : namely, 5 
drops of a 1 per cent, gold chlorid solution in 10 c.c. of 
distilled water (to which it is sometimes advisable to add 2 
or 3 drops of glacial acetic acid), for ten minutes. 

11. Removal of any unreduced silver by placing the sec- 
tions in a 5 per cent, solution of hypophosphite of sodium 
(to which 1 to 2 drops of a saturated solution of sodium 
sulphite are sometimes added) for one-half to one minute. 

12. Wash thoroughly in water. 

13. Dehydrate quickly in absolute alcohol : xylol; balsam. 
Bielschowsky has recently modified his method as follows 

for staining tissues in mass before cutting, but it is not so 
reliable as the stain for frozen sections : 

1. Fix in formaldehyde. 

2. Wash in water. 

3. Place in pure pyridin for three to four days. 

4. Wash thoroughly in distilled water. 

5. Place in a 3 per cent, solution of silver nitrate for three 
to five days. 

6. Wash quickly in water. 

7. Place in the second silver bath (step 5 in directions 
above) diluted to 86 c.c. for twenty-four hours. 



134 PATHOLOGICAL TECHNIQUE. 

8. Wash in water. 

9. Reduce in formaldehyde. 

10. Dehydrate in graded alcohols ; clear and imbed in 
paraffin ; cut sections. 

11. Xylol, balsam. 

The axis cylinders and nerve-fibrils are stained black. 

4. Stroebe's Aniline-blue Stain for Nerve -fibers in Hard- 
ened Sections. — Harden tissues in M tiller's fluid. I. Stain 
one-half to one hour in a saturated aqueous solution of 
aniline-blue. 

2. Wash in water. 

3. Transfer to a small dish of alcohol to which are added 
20 to 30 drops of a 1 per cent, alcoholic solution of caustic 
potash (caustic potash 1 to alcohol 100 : let the mixture stand 
for twenty-four hours ; then filter). In one to several minutes 
the sections become bright brownish-red and transparent. 

4. Transfer to distilled water for five minutes. The sec- 
tion becomes bright blue again. 

5. Stain in a half-saturated aqueous solution of safranin, 
one-quarter to one-half hour long. 

6. Wash out and dehydrate in absolute alcohol. 

7. Xylol, xylol balsam. 

5. Chlorid-of-iron and dinitroresorcin method for the study 
of degenerated peripheral nerves : 

1. Place fresh pieces of peripheral nerves for several days 
in a solution of — 

Chlorid of iron, 1 part ; 

Distilled water, 4 parts. 

2. Wash out thoroughly in water. 

3. Transfer to a saturated solution of dinitroresorcin in 75 
per cent, alcohol for several weeks. 

4. Wash, dehydrate, imbed, etc. 

A permanent green color is formed which stains the nerves 
green and brings out the green axis-cylinders very sharply. 

The stain will succeed with preparations which have been 
hardened in Flemming's solution or Miiller's fluid. 

Golgi's methods are sometimes employed for the study of 
the terminal processes of nerve-fibers (for directions see p. 125). 



HISTOLOGICAL METHODS. 135 

Stains for the Myelin-sheath.— The myelin-sheath 
of nerve-fibers is a form of fat, and like it possesses the 
property of reducing osmic acid, by means of which a selec- 
tive sheath stain can be obtained. Unfortunately, however, 
the osmic acid penetrates to but a very slight depth. Three 
methods employing osmic acid are given, but they are all 
expensive and not so satisfactory as those employing hema- 
toxylin. 

The differential hematoxylin stain, originated by Weigert, 
and ordinarily used, depends on some chemical reaction 
which takes place between the myelin and a chrome salt, in 
consequence of which the myelin is fixed so that it will not 
later be dissolved out by alcohol or ether, and at the same 
time is so mordanted that it can be deeply stained with 
hematoxylin, to which it clings when treated with certain 
decolorizers. This reaction between the myelin and the 
chrome salts in general use takes place very slowly at the 
ordinary temperature ; six weeks to several months are 
usually required. Weigert's latest method depends on the 
interaction of two chrome salts in the same solution, in 
consequence of which the time needed for this reaction or 
mordanting is reduced to four days. The solution may be 
used alone, but is best combined with formaldehyde or used 
after it. 

A. Weigert's Myelin-sheath Stain. — In this method five 
steps are involved, but the first two can be, and often are, 
combined in one. These five steps are fixation, primary 
mordanting, secondary mordanting, staining, and differentia- 
tion. These different steps will be considered separately. 

1. Fixation. — Place the tissues in a 4 per cent, solution of 
formaldehyde (10 per cent, solution of formalin) for four 
days to several weeks or indefinitely, using several times the 
volume of the tissue. Change the solution at the end of 
twenty-four hours, and thereafter whenever it becomes cloudy. 
Large masses of nervous tissue, like the medulla and pons 
or the basal ganglia, should be fixed in formaldehyde for 
one to three weeks. 

2. Primary Mordanting. — Cut the tissues fixed in formal- 



136 PATHOLOGICAL TECHNIQUE. 

dehyde into slices not over 1 cm. thick, and place in the 
following solution for four to five days at room -temperature : 

Bichromate of potassium, 5 ; 

Fluorchrom, 2 ; 

Water, ad 100. 

Steps 1 and 2 may be combined by adding 4 per cent, of 
formaldehyde to the mordanting solution and placing the 
fresh tissues directly in the mixture. 

3. Secondary Mordanting. — Transfer the tissues to the 
following solution for twenty-four to forty-eight hours : 

Acetate of copper, 5.0; 

Acetic acid, 36 per cent, solution, 5.0; 

Fluorchrom, 2.5 ; 

Water, ad 100.0 

(For method of preparation see page 70.) 

Weigert always transfers blocks of tissue to the secondary 
mordant, of which the function is to intensify the staining 
reaction. Many workers, however, prefer to employ the 
secondary mordant on sections only. In this case they 
place the tissues directly from the first mordant into graded 
alcohols, imbed in celloidin, and cut sections before step 3. 
Either way gives good results, but the first is the simpler. 

Weigert also recommends the following iron solution as a 
secondary mordant, but if applied to blocks of tissue, they 
must be well washed in running water before being dehy- 
drated and imbedded in celloidin, because otherwise the 
iron will rust the knife badly. Of course, if this mordant is 
applied to sections only, prolonged washing is not necessary : 

Iron alum (ammonioferric alum), 5 ; 

Acetic acid, 5 ; 

Water, ad 100. 

After the secondary mordant the tissue is dehydrated in 
graded alcohols, imbedded in celloidin, and sections cut in 
the usual manner. 

4. Staining. — Stain sections in the following solution for 
twelve to twenty-four hours : 



HIS TOL O GICA L ME THODS. 



137 



Ripened 10 per cent, solution of hematoxylin in 

absolute alcohol, 10; 

Saturated aqueous solution of carbonate of 

lithium, 1 ; 

Water, 90. 

Keep on hand as a stock solution a 10 per cent, solution 
of hematoxylin in absolute alcohol. At least ten days of 
exposure to light are required to ripen this solution so that 
it can be used for staining. Combine with the carbonate of 
lithium and the water at the time of using in such propor- 
tions as are wanted for immediate use. Wash the sections 
thoroughly in water before differentiation. 

Another staining solution which Weigert recommends 
highly is the following — it consists of two parts : 

(a) Liquor ferri sesquichlorati (officinal), 4 c.c. ; 

Water, 96 " 

{b) Ripened 10 per cent, solution of hema- 
toxylin in absolute alcohol, 10 " 

96 per cent, alcohol, 90 " 

Mix thoroughly equal parts of these two solutions just 
before using, and pour over the sections. Stain overnight 
or longer at room-temperature. Pour off solution and wash 
with water. 

5. Differentiation. — The sections are differentiated in the 
following solution which it is sometimes advisable to dilute 
with water : 



Borax, 

Ferricyanid of potassium, 

Water. 



2.0; 

2.5; 
I OO.O. 



After the first staining method given above the decolor- 
ized tissues appear yellow ; after the iron-hematoxylin solu- 
tion they are colorless. Moreover, this latter method stains 
the very finest fibers and at the same time the coarse fibers, 
such as occur, for example, in the nerve-roots. 

After differentiation the sections should be thoroughly 
washed in water, dehydrated in alcohol, cleared in the anilin 



138 PATHOLOGICAL TECHNIQUE. 

oil-xylol or carbol-xylol mixture, and mounted in xylol 
balsam. 

The steps of the process may be summed up as follows : 

1. Fix in 10 per cent, formaldehyde four days or longer. 

2. Mordant in the bichromate of potassium-fluorchrom 
solution four to five days. 

3. Mordant in the acetate of copper-fluorchrom solution 
twenty-four to forty-eight hours, or in the iron solution 
twenty-four to forty-eight hours. 

4. Dehydrate in graded alcohols. 

5. Imbed in celloidin. 

6. Stain sections in the alcoholic hematoxylin solution or in 
the iron-hematoxylin solution for twelve to twenty -four hours. 

7. Wash off in water. 

8. Differentiate in the borax-ferricyanid of potassium 
solution. 

9. Wash thoroughly in water. 

10. Dehydrate in alcohol. 

11. Clear in the anilin oil-xylol or carbol-xylol mixture. 

12. Mount in xylol balsam. 

B. Pal's Modification of Weigert's Myelin-sheath Stain. 
— 1. Fixation and primary mordanting as for Weigert's 
method. 

2. Place sections for several hours in a ^ per cent, aqueous 
solution of chromic acid, or for a longer time in a 2-3 per 
cent, solution of bichromate of potassium. This step is 
often omitted, especially when the tissues have been but 
recently mordanted. 

3. Transfer sections to Weigert's alcoholic hematoxylin 
solution for twenty-four to forty-eight hours (if necessary 
for an hour in the incubator at 37 C). This solution is as 
follows : 

Ripened 10 per cent, solution of hematoxylin 

in absolute alcohol, 10. 

Water, 90. 

4. Wash in water plus 1 to 3 per cent, of a saturated 
aqueous solution of carbonate of lithium until the sections 
appear of a uniform deep-blue color. 



HISTOLOGICAL METHODS. 139 

5. Differentiate for twenty seconds to five minutes in a \ 
per cent, aqueous solution of permanganate of potassium 
until the gray matter looks brownish-yellow. 

6. Transfer to the following solution : 

Oxalic acid, I ; 

Sulphite of potassium, 1 ; 

Water, 200. 

for a few seconds until the gray matter is colorless or 
nearly so. 

7. Wash thoroughly in water. 

8. Dehydrate in 95 per cent, alcohol. 

9. Oil, xylol balsam. 

Steps 5 and 6 sometimes have to be repeated when the 
differentiation has not been complete. 

Of all the numerous modifications of Weigert's original 
myelin-sheath stain, the only one that has found general 
acceptance until recently is Pal's. It has the following ad- 
vantages : It gives very clear pictures ; everything except the 
sheaths is completely decolorized, so that contrast-stains are 
possible; it is more successful with thick sections than Weigert's 
method ; the separate steps are quicker. On the other 
hand, the danger of decolorizing the sheaths of the finer 
fibers is greater. 

C. Kulschitzky-Wolter's Modification of Weigert's 
Myelin-sheath Stain. — It is claimed to stain the myelin 
sheaths intensely and with great certainty. 

1. Fix in formaldehyde, mordant in Weigert's chromalum 
mixture, dehydrate in alcohol, imbed in celloidin. 

2. Stain sections twenty-four hours at a temperature of 
37 C in the following solution well ripened : 

Hematoxylin, 1 gram; 

Absolute alcohol, 10 c.c. 

2 per cent, acetic acid, 90 " 

3. Dip sections in Miiller's fluid. 

4. Differentiate in a | per cent, aqueous solution of per- 
manganate of potassium. 

5. Wash in water. 



140 PATHOLOGICAL TECHNIQUE. 

6. Place in Pal's oxalic acid solution until the gray matter 
is colorless. 

7. Wash thoroughly in tap water to which some sal am- 
moniac is added. 

8. Dehydrate in alcohol. 

9. Clear and mount in balsam. 

D. Exner's Stain. — The tissue should be obtained as 
soon as possible after death, although the method will 
succeed with tissues even over twelve hours old. 

1. Place pieces of brain or cord not over \ cm. thick in a 
I per cent, aqueous solution of osmic acid, using at least 
ten times as much fluid as the volume of the specimen. 

2. Change the osmic-acid solution on the second day. 

3. After five or six days wash thoroughly in water. 

4. Dehydrate, imbed, etc. 

5. Examine sections in glycerin rendered slightly ammo- 
niacal. 

The myelin-sheaths appear gray to black. The prepara- 
tions are not permanent. 

This procedure has been almost entirely replaced by Wei- 
gert's method, which has numerous advantages. Lately, 
however, it has been brought forward again by Heller, who 
uses a photographic developer to reduce the osmic acid and 
to make possible permanent mounts. He has lately pub- 
lished the following method for sections, but it cannot be 
unconditionally recommended : 

E. Heller's Myelin-sheath Stain. — 1. Harden as for the 
Weigert method (Heller used Miiller's fluid). 

2. Imbed in celloidin. 

3. Place sections in a 1 per cent aqueous solution of os- 
mic acid for ten minutes in thermostat or for half an hour 
at room-temperature. 

4. Wash in water. 

5. Reduce in the following developer: 

Sulphate of sodium, J25 ; 

Carbonate of sodium, 70; 

Water, 500 ; 

Pyrogallic acid, 15. 



HISTOLOGICAL METHODS. 141 

6. Wash in water. 

7. Differentiate in an aqueous solution of permanganate 
of potassium, \ per cent, or less. 

8. Remove the brown of the permanganate of potassium 
In a 1 per cent, aqueous solution of oxalic acid. 

9. Wash in water. 

10. Alcohol, oil, chloroform balsam. 

F. Robertson's Modification of Heller's Myelin-sheath 
Stain. — 1. Harden in Weigert's fluorchrom-copper solution 
plus 4 per cent, of formaldehyde ; in other words, use the 
mordant for neuroglia-fibers (page 144) eight to ten days. 

2. Wash off in water. 

3. Alcohol ; imbed in celloidin. 

4. Stain sections in a 1 per cent, solution of osmic acid 
half an hour in the dark. 

5. Place in a 5 per cent, aqueous solution of pyrogallic 
acid for half an hour. 

6. Differentiate in a \ per cent, aqueous solution of per- 
manganate of potassium one to four minutes. 

7. Remove brown color in 1 per cent, oxalic acid three to 
five minutes. 

8. Alcohol, oil, balsam. 

It is important to wash carefully in water between each 
of the staining steps. 

G. Myelin-sheath Stain for Frozen Sections (Wright) . — 
I. Fix in 4 per cent, formaldehyde solution. 

2. Cut frozen sections. 

3. Place in 50 per cent, alcohol for one minute, moving 
the section about in the fluid. 

4. Place in 10 per cent, aqueous solution of ferric chloride 
for one minute. 

5. Without washing transfer the section to a small quan- 
tity of a freshly prepared aqueous solution of hematoxylin 
for five minutes or longer. This is conveniently prepared 
by placing in a test-tube three or four small crystals of hem- 
atoxylin and 10 c.c. of distilled water, and heating over a 
flame until the solution is complete. 

6. Wash quickly in water. 



I4 2 PATHOLOGICAL TECHNIQUE. 

7. Differentiate by moving the section about in 10 per 
cent, aqueous solution of ferric chloride until the gray sub- 
stance is well defined and the connective tissue of the pia 
mater appears yellow. Care should be taken not to differ- 
entiate too much and thus decolorize the myelin sheaths. 

8. Wash thoroughly in a large quantity of distilled water 
or further decolorization will take place. 

9. Dehydrate in alcohol. 

10. Clear in origanum oil. 

1 1. Press the section flat on the slide with blotting paper, 
or a pad of folded filter paper, and mount in xylol balsam. 

Stains for Neuroglia-fibers. — It is possible to obtain 
a differential stain of the neuroglia-fibers in man by several 
different methods. The tissue must be as fresh as pos- 
sible. The best results are obtained with tissues placed 
in the fixing solution within one hour after death. After 
four to six hours the results are only fair ; after twenty-four 
hours they are practically nil. The peculiar property in the 
neuroglia-fibers on which the differential stain depends has 
undergone some chemical change or has disappeared. It is 
retained longest where the fibers are most numerous, as 
about the central canal. 

Zenker's fluid, formaldehyde, or alcohol may be used as a 
fixative, according to the staining method preferred. Fixa- 
tion in Zenker's fluid, followed by staining with phospho- 
tungstic-acid hematoxylin, can be highly recommended and 
is much the quickest and simplest method. The Weigert 
method given here, and the only one he ever published, had 
been discarded by him in favor of a newer and better method, 
but, unfortunately, the secret of it perished with him. 

For all the methods given it is imperative that the tissue 
should be cut into thin slices, not over 2 to 3 mm. thick, 
before it is placed in the fixing solution used. 

No one of these methods stains neuroglia fibrils only. All 
stain fibrin, and all, with the possible exception of Weigert's, 
stain fibroglia and myoglia fibrils when these are freshly 
fixed. 



HISTOLOGICAL METHODS. 143 

A. Mallory's Phosphotungstic-acid Hematoxylin Stain : 

1. Fixation in Zenker's fluid, 24 hours. 

2. Running water, 24 

3. Alcohol, 80 per cent, 24 " 

4. Paraffin or celloidin imbedding. 

5. Treat sections with iodin solution (Gram's iodin solu- 
tion or a J per cent, alcoholic solution) to remove the mer- 
cury precipitate, five to ten minutes. 

6. Alcohol, 95 per cent., several changes to remove 
iodin. 

7. Water. 

8. Permanganate of potassium, \ per cent, aqueous solu- 
tion, for three to five minutes, sometimes ten to twenty- 
minutes. 

9. Wash in water. 

10. Oxalic acid, 5 per cent, aqueous solution, five to ten 
minutes, sometimes longer. 

1 1 . Wash thoroughly in several changes of water. 

1 2. Stain in phosphotungstic-acid hematoxylin for twelve 
to twenty-four hours. 

13. Transfer directly to 95 per cent, alcohol, followed by 
absolute alcohol for paraffin sections, and dehydrate quickly. 

14. Clear in xylol (filter-paper blotting method for celloidin 
sections) and mount in xylol balsam. 

Neuroglia, fibroglia, and myoglia fibrils and fibrin blue, 
collagen fibrils reddish-brown ; the coarse elastic fibrils some- 
times stain of a purplish tint. 

In step 1 3 the treatment with alcohol should not be pro- 
longed over one to two minutes ordinarily, as the alcohol 
extracts the reddish color and destroys the sharp contrast 
between the different kinds of fibrils. 

If after step 1 2 the sections are placed in a strong alcoholic 
solution (10 to 20 per cent.) of chlorid of iron for one to sev- 
eral minutes, followed by thorough washing in water, the 
collagen-fibrils and other reddish-stained structures are com- 
pletely decolorized. 

Xylol must be used as the clearing reagent, because after 
origanum and other oils the blue color fades. 



144 PATHOLOGICAL TECHNIQUE. 

Sections stained in phosphotungstic-acid hematoxylin keep 
for years if not unduly exposed to the light. 

This same staining method can be used after formaldehyde 
fixation if the tissues are first carried through Zenker's fluid 
in the ordinary way, just as if the tissue were perfectly fresh. 

B. Weigert's Differential Stain for Neurog-lia-fibers. — 
A. Fix thin pieces of tissue, not over \ cm. thick, in a 4 per 
cent, solution of formaldehyde for at least four days. 

B. Mordant in the following solution for four to five days 
in the incubator or for eight days at room-temperature : 

Acetate of copper, 5 gr. ; 

Acetic acid, 36 per cent, solution, 5 c.c. ; 

Fluorchrom, 2.5 gr.; 

Water, ad 100 c.c. 

Boil the fluorchrom and water in a covered dish, turn off the 
gas, add the acetic acid and then the" acetate of copper ; stir 
briskly until the latter is dissolved, then cool. The solution 
remains clear. 

(Steps 1 and 2 may be combined by adding 4 per cent, of 
formaldehyde to the above solution ; change on the second 
day ; harden eight days.) 

C. Wash off in water ; dehydrate in alcohol ; imbed in 
celloidin. 

D. Reduction of copper salt in sections : 

1. Place* 5 the sections, which must not be over .02 mm. 
thick, in a -J- per cent, aqueous solution of permanganate of 
potassium for ten minutes. 

2. Wash off with water. 

3. Decolorize and reduce for two to four hours in the fol- 
lowing solution, carefully filtered : 

Chromogen, 5 gr. ; 

Formic acid (sp. gr. 1.20), 5 c.c; 

Water, ad 100 " 

to 90 c.c. of which are added just before using 10 c.c. of a 
10 per cent, solution of sulphite of sodium. 

The sections lose their color in a few minutes, but are best 
kept in, the solution as long as above directed. 



HISTOLOGICAL METHODS. 1 45 

The sections can now be stained in the manner to be de- 
scribed, but the color of the fibers will be more intense if the 
following steps are added, and a slight yellowish contrast- 
stain is obtained for the ganglion and ependymal cells and 
for the larger nerve-fibers. This step has one disadvantage, 
however : the connective-tissue fibers stain blue after it. 

E. Further reduction of copper salt : 

1. Wash twice in water. 

2. Place sections in a carefully filtered saturated (5 per 
cent.) aqueous solution of chromogen overnight. 

3. Wash in water. 

4. The sections are now ready for staining or may be pre- 
served until wanted in — 

80 per cent, alcohol, 90 c.c. 

5 per cent, oxalic acid, 10 " 

F. Staining of neuroglia-fibers : 

1. Lift section from large dish of water on slide freshly 
cleaned with alcohol ; blot with filter-paper (method recom- 
mended by Weigert for attaching sections to slide). 

2. Stain in the following mixture : 

Saturated solution of methyl-violet in 

70-80 per cent, alcohol, 100 c.c; 

(saturated with aid of heat ; decanted 
when cold). 

5 per cent, aqueous solution of oxalic 

acid, 5 " 

The oxalic acid is added to render the preparations more 
lasting. The staining is practically instantaneous. 

3. Wash off with normal salt solution. 

4. Iodin solution : 5 per cent, iodid-of-potassium solution 
saturated with iodin. It is simply poured on and then off, 
as the reaction is instantaneous. 

5. Wash off with water and blot with filter-paper. 

6. Decolorize thoroughly in equal parts of xylol and 

aniline oil. 
10 



146 PATHOLOGICAL TECHNIQUE. 

7. Wash repeatedly with xylol or the stain will not keep. 

8. Canada balsam. 

The sections keep better if exposed for from two to five 
days to diffuse light before being put away. 

C. Benda's Stain for Neuroglia Fibrils. — Hardening. — 
I. Fix fresh material for at least two days in 90 to 93 per 
cent, alcohol. 

2. Place thin sections (not over 5 mm. thick) in 10 per 
cent, nitric acid for twenty-four hours. 

3. Two per cent, aqueous solution of bichromate of potas- 
sium, twenty-four hours. 

4. One per cent, aqueous solution of chromic acid, forty- 
eight hours. 

5. Wash in running water for twenty-four hours, harden in 
graded alcohols, imbed in paraffin. 

Staining. — Iron-alizarin-toluidin-blue Stain. — 1 . Mordant 
the sections for twenty-four hours in a 4 per cent, solution 
of iron-alum. 

2. Wash off in running water. 

3. Twenty-four hours in dilute amber-yellow aqueous 
solution of sodium sulphalizarate. 

4. Dip in water and blot with filter-paper. 

5. Stain in a 1 per cent, aqueous solution of toluidin-blue, 
warm until steam rises, then let it stain about fifteen minutes 
in the cooling fluid; or stain one to twenty-four hours in 
cold toluidin-blue. 

6. Dip in 10 per cent, acetic acid or in very dilute picric 
acid. 

7. Dry with filter-paper and dip in absolute alcohol. 

8. Differentiate in beech creasote about 10 minutes, con- 
trolling result with microscope. 

9. Dry with filter-paper ; xylol balsam. 
Degenerations of the Nervous System. — The same 

methods apply to the study of degenerations in nervous tis- 
sue that apply elsewhere, except in the demonstration of 
fat. Both myelin and fat reduce osmic acid, so that the 
ordinary test for fat in the hardened tissues fails. Marchi 
and Algeri, however, have shown that after myelin has been 



HISTOLOGICAL METHODS. 147 

mordanted for eight days or over in Muller's fluid or other 
solution of the bichromates, it loses the property of reduc- 
ing the osmic acid, while fat retains the property unimpaired. 
On this peculiarity is based their method for differentiating 
fat from myelin. 

Marchi and Algeri's Method for Staining- Fatty De- 
generated Myelin-sheaths of Nerve-fibers. — 1. Harden in 
Muller's fluid or in formaldehyde, followed by Muller's fluid, 
for eight days to three months. 

2. Transfer tissue for five to eight days directly into the 
following solution : 

Muller's fluid, 2 parts ; 

I per cent, osmic-acid solution, 1 part. 

3. Wash out thoroughly in water. 

4. Dehydrate in alcohol. 

5. Imbed in celloidin. 

6. Clear in chloroform and mount in properly prepared 
chloroform balsam (see page 106). 

METHODS OF FIXING AND EXAMINING SPECIAL 
ORGANS AND TISSUES. 

Tissues which are to be hardened should be obtained as 
fresh as possible. For this reason autopsies rarely furnish 
such perfect material as is obtainable from experimental 
lesions in animals or from surgical operations. Still, most 
of the pathological material comes from autopsies, and it is 
encouraging to know that very good work can often be done 
with tissues not fixed until twenty-four hours or even more 
after death. The most valuable autopsies are those which 
are freshest, and in which but one etiological factor has been 
concerned, so that the relation between the cause and the 
lesion produced is uncomplicated and can be readily grasped 
and understood. 

The choice of the proper fixing reagent varies with the 
tissue, the lesion, and the use to which the material is to be 
put. It is advised as a routine procedure to preserve tissues 



148 PATHOLOGICAL TECHNIQUE. 

in two fluids : in Zenker's fluid for general histological study, 
including both the injurious agents of all sorts and the in- 
flammatory reactions to them ; in formaldehyde for the preser- 
vation of fat and myelin, and certain substances to which it 
may be desirable to apply chemical tests. With these two 
fixatives properly applied it is possible to go a long way in 
pathological histology. Orth's fluid may be substituted for 
Zenker's fluid, but is distinctly not so good. Alcohol is re- 
quired for the preservation of certain substances, such as 
glycogen, urate of sodium crystals, and hemosiderin, and cor- 
rosive sublimate fixation is necessary for certain special stains 
for mucin. 

It is imperative that pieces of tissue for histological study 
should be placed in the proper fixative as soon after the re- 
moval of the organs from the body as possible, so that the 
surface will not dry or the blood and other fluids escape 
from the vessels. Do not wash off the surface with water. 
The tissues should almost invariably be cut into thin slices, 
not over 2 to 4 mm. thick. 

In preserving tissues it is very important to use enough of 
the fixing reagent — ten to fifteen times as much as there is 
tissue. It is advised to harden tissue in flat-bottomed glass 
dishes and to stir them occasionally, so that they may come 
in contact with fresh fluid. 

After Zenker fixation the best stain to use for general his- 
tological study is methylene-blue and eosin. For class use 
alum hematoxylin and eosin make a fairly satisfactory sub- 
stitute, but do not demonstrate any bacteria present. The 
other useful stains are phosphotungstic acid hematoxylin, 
the aniline blue method for collagen fibrils, and VerhoefTs 
elastic tissue stain. After formaldehyde fixation the most 
interesting results are obtained by staining frozen sections 
with Scharlach R and alum hematoxylin. 

These methods of fixation and staining are applicable to 
most of the tissues listed below, and constitute the routine 
stains for almost all organs outside of the central nervous 
system, and even there they are often useful. 

Acute Inflammatory Exudations; Granulation- 
tissue. — The elements in acute inflammatory exudations 



HISTOLOGICAL METHODS. 1 49 

which require preservation are chiefly leucocytes of different 
sorts, serum, fibrin, and red blood-corpuscles. The best 
general fixative for them all is Zenker's fluid. It not only 
preserves perfectly the characteristic nuclei of the leucocytes, 
but also the cytoplasm, which stands out sharply in contrast- 
staining with eosin. The albumin of the serum is coagulated 
into a finely granular material. The fibrin and red blood- 
corpuscles stain brilliantly with eosin. 

I/ling". — In the preservation of the lungs it is important 
to save portions that have not been squeezed, so that the re- 
lations of the exudations may not have been changed or the 
alveoli compressed. Thin slices are usually preferable to 
cubical pieces, and should be cut with a very sharp knife, so 
as not to compress the tissue, and dropped immediately into 
the fixing fluid, before the contents of the bronchi and of 
small cavities have had time to run out. An emphysematous 
lung is so delicate that it is usually better to inject a whole 
lung through the bronchi with the fixing fluid or to snip out 
small pieces with scissors. Zenker's fluid and formaldehyde 
are the most useful fixatives. 

Bone-marrow and Spleen. — On account of the simi- 
larity in the cellular content of these organs they are con- 
sidered together. They may be studied by both smear and 
section methods. 

Sections. — The pieces of tissue to be fixed should be 
about 2 mm. thick. The sections are to be cut in paraffin 
and are to be as thin as possible. For general purposes, 
fixation in Zenker's fluid and staining by the eosin-meth.y- 
lene-blue method are recommended. Other recommenda- 
tions are : 

1. For the study of erythroblasts and the formation of 
red blood-corpuscles : fixation in corrosive sublimate and 
staining by eosin soluble in alcohol and alkaline methylene- 
blue. 

2. For the demonstration of the granules of myelocytes 
and leucocytes : fixation in corrosive sublimate and staining 
by Wright's blood-staining fluid undiluted, then washing in 
water, dehydrating in acetone, clearing in oil of turpentine, 



150 PATHOLOGICAL TECHNIQUE. 

and mounting in turpentine colophonium . The Biondi- 
Heidenhain triple stain may also be used. 

Schridde's Method of Staining the Granulations of 
Myelocytes and Leucocytes in Sections. — The tissue is 
best fixed in Orth's fixing fluid, but other fixatives may be 
used. The sections should not be thicker than 5 ,u and 
should be fixed to the slide with Mayer's albumen mixture. 
They are stained for twenty minutes in Giemsa's stain, diluted 
with distilled water in the proportion of two drops of the stain 
to each cubic centimeter of water. The mixture must be 
freshly made before using. When the staining is completed 
the preparation is washed in water, the excess of water re- 
moved with filter-paper, and the section immediately placed 
in pure acetone. If the acetone extracts color from the 
preparation, it is impure and should not be used. The sec- 
tion is then cleared with toluol or xylol and embedded in 
neutral xylol balsam. Care should be taken not to allow 
the sections to become dry from the rapid evaporation of the 
acetone. 

The neutrophile granulations are stained a violet-red ; the 
eosin granulations red ; the granulations of the mast-cells 
dark blue, and the granulations in the cytoplasm of the 
megakaryocytes violet-red. All nuclei are blue and the red 
blood-corpuscles grass-green. The connective tissue is of a 
pale red color. 

Wright's Method for the Differential Staining of the 
Blood-platelets and the Giant Cells (Megakaryocytes) of 
the Bone-marrow. — The tissue should be absolutely fresh. 
It is fixed in a 4 per cent, aqueous solution of formaldehyde 
or in a saturated solution of corrosive sublimate in 0.9 per 
cent, salt solution. Tissue that has been decalcified is not 
suitable. The sections are cut in paraffin and should not be 
more than 7 microns thick. They are stained while affixed 
to the slide by Mayer's egg-albumen method. 

The staining fluid and the mode of its preparation are 
described below. 

The staining, clearing, and mounting are carried out as 
follows : 



HISTOLOGICAL METHODS. I 5 I 

I. Equal parts of the staining fluid and distilled water are 
mixed in a small wineglass and immediately poured on to 
the slide. The measuring is conveniently done by means of 
a small pipette provided with a rubber bulb. At least 2 c.c. 
of the freshly diluted staining fluid are thus spread out over 
the slide, which should be supported upon some object in 
such a way as to prevent the fluid from running off. The 
spreading out of the fluid in a layer is important, because it 
facilitates the evaporation of the alcohol, whereby the stain- 
ing elements slowly precipitate out of solution and, while 
doing so, stain the tissue elements. This precipitate appears 
as a yellowish, metallic scum which slowly forms on the sur- 
face of the mixture. The diluted staining fluid is allowed to 
act for about fifteen minutes, when the preparation is imme- 
diately washed in water. The exact time required for the 
best results has to be determined for each batch of the stain- 
ing fluid. The proper staining of the preparation may be 
judged by examining it under a low magnifying power by 
artificial light after pouring back the diluted staining fluid 
into the wineglass. The staining is stopped by washing the 
preparation in water, when the cytoplasm of the giant-cells 
has acquired a bright red color and the fibrils of the reticulum 
begin to take on a red color also. If the staining is found 
not sufficiently intense, the diluted staining fluid is poured 
back on the preparation and allowed to act longer. Over- 
staining and the formation of a black-red granular precipitate 
on the preparation occur if the diluted staining fluid is allowed 
to act longer than a certain time. 

2. Dehydrate in pure acetone. 

On account of the great volatility of acetone, some care 
is necessary to prevent the drying of the preparation, which 
should be avoided. 

3. Clear in xylol or pure oil of turpentine. 

4. Mount in a thick solution of colophonium in xylol or 
pure oil of turpentine. 

Before mounting the preparation the superfluous turpen- 
tine should be removed, because this reagent rapidly takes 
up water from the air, and thus may cause the clouding of 
the preparation or the fading of the stain. 



152 PATHOLOGICAL TECHNIQUE. 

The solution of colophonium is made by saturating a 
quantity of turpentine with powdered colophonium, and 
keeping the filtered solution in the paraffin embedding oven 
until it has evaporated to the required consistence. 

The use of acetone instead of alcohol for dehydrating is an 
important feature of the method, for the latter spoils the 
characteristic staining of the granules in the giant-cells and 
platelets. 

The staining fluid is composed of I part of a modified 
methylene-blue solution and io parts of an o.i per cent, so- 
lution of water-soluble eosin in pure methyl alcohol. 

The solution of methylene-blue is prepared as follows ; 
One gram of methylene-blue "med. pur." is dissolved as 
thoroughly as possible in ioo ex. of an 0.5 per cent, aqueous 
solution of sodium bicarbonate in an Erlenmeyer flask. The 
flask and its contents are then placed in an ordinary steam 
sterilizer and kept at ioo° C. for one hour and a half, count- 
ing the time after the steaming has become vigorous. When 
cool, the mixture is filtered and the filtrate is the modified 
blue solution. It must be of a well-marked purple color 
when viewed in a thin layer by the yellow transmitted light 
of an ordinary incandescent electric bulb. This color appears 
only after cooling. 

Variations in the solutions of the blue and of the eosin 
may require that the proportions above given be changed 
slightly. An excess of eosin delays the appearance of the 
scum on the surface of the diluted staining fluid, and pro- 
longs the time required for staining. On the other hand, an 
excess of the modified blue component hastens the appear- 
ance of the scum, and may cause overstaining and the gran- 
ular precipitate to form on the preparation. 

The blood-platelets typically appear as rounded bodies, 
more or less jagged in outline, and composed of a hyaline, 
blue-staining substance, in which are embedded, chiefly in the 
central portions, fine red to purplish granules. The cyto- 
plasm of the giant-cells shows the same structure and stain- 
ing peculiarities. The sections should be examined by- an 
incandescent electric light in order to bring out the colors to 



HISTOLOGICAL METHODS. I 53 

the best advantage. By this method all grades of transition 
can be shown between pseudopod-like processes of the giant- 
cells or detached masses of giant-cell cytoplasm and blood- 
platelets. 

Smear preparations may be made either upon slides or 
cover-glasses, and stained by Wright's blood-stain, as in the 
case of blood-smears. For the best results the preparation 
should not be allowed to dry, but should be stained imme- 
diately while still wet, and, after staining, dehydrated with ab- 
solute alcohol, cleared with xylol, and mounted in balsam in 
the same manner as a section affixed to the slide. A longer 
period of staining than that directed for blood-smears is 
usually desirable. 

Special Methods for Smear Preparations from Bone- 
marrow. — Thoroughly tease a small bit of the marrow in a 
few drops of blood-serum and from this mixture prepare the 
smear preparation, as in the case of a blood-smear. The 
preparation, however, must not be allowed to dry, but is fixed 
immediately by methyl-alcohol (one minute) while still wet. 
It is then covered for three to five minutes with a mixture 
of equal parts of Wright's blood-stain and distilled water. 
This mixture must have been prepared immediately before 
use. After staining, the preparation is not allowed to dry, 
but is washed in water, dehydrated with acetone, cleared 
with oil of turpentine, and mounted in turpentine colopho- 
nium. Instead of methyl-alcohol, corrosive sublimate, Zen- 
ker's fluid, or a I per cent, solution of osmic acid may be 
used for fixation, each being allowed to act about one min- 
ute, when the preparation is to be washed in water, covered 
with 95 per cent, alcohol for one minute, rinsed in water, and 
then treated as indicated. It is important that the prepara- 
tion be not allowed to dry at any stage of the process. 

By these methods many of the finer details of the marrow- 
cells are brought out much better than by the usual smear 
method. 

Kidney. — Zenker's fluid and formaldehyde as fixatives 
answer most purposes, but alcohol is required to preserve 
glycogen and certain crystalline deposits. The Scharlach R. 



154 PATHOLOGICAL TECHNIQUE. 

stain, after formaldehyde fixation, has to a large extent re- 
placed Flemming's solution. Fixation by boiling is still 
used to demonstrate an albuminous exudate in the capsular 
space. The general staining methods already recommended 
will be found the most satisfactory. 

In cases of chronic nephritis the capsule should not be 
peeled from those parts kept for microscopical purposes. 

Paraffin embedding is generally to be preferred for 
the kidney, especially when lesions of the glomeruli are 
present. 

Gastro -intestinal Tract. — Portions of the stomach or 
intestine should be hardened as soon after death as possible 
for satisfactory study, because the gastro-intestinal tract so 
rapidly undergoes post-mortem changes. It has been recom- 
mended in appropriate cases, where an autopsy is allowable, 
to inject the stomach with the desired fixing solution by 
means of a rubber tube as soon after death as is permissible. 
Under no circumstances should the surface of the intestine 
or stomach be washed with water. Use either normal salt 
solution or some of the fixing solution. It is important to 
keep the tissue flat while hardening. This can usually be 
done by laying it with the peritoneal surface down on thick 
filter-paper, to which it readily sticks. Sometimes it is 
necessary to pin the specimens down at the edges on flat 
pieces of cork. Do not let the surface dry before the speci- 
men is placed in the fixing solution. Zenker's fluid can be 
highly recommended as a fixative, but alcohol is sometimes 
to be preferred. 

I4ver. — Fat is most easily and satisfactorily demonstrated 
by the Scharlach R. stain after formaldehyde fixation. Ne- 
crosis of liver-cells is best shown by the eosin-methylene- 
blue stain after fixation in Zenker's fluid. The necrotic cells 
stand out of a deep pink color in sharp contrast to the other 
cells. The aniline blue stain is especially useful in the study 
of the lesions associated with chronic passive congestion and 
with amyloid deposit. 

For obtaining the iron reaction with hemosiderin in cases 
of pernicious anemia, and for the reactions of amyloid, harden 
in alcohol or formaldehyde. 



HISTOLOGICAL METHODS. 1 55 

For general histological study Zenker's fluid will be found 
exceedingly useful. 

The bile-capillaries may be demonstrated by the same 
method that is used for neuroglia fibrils — namely, fixation in 
Zenker's fluid, following by staining in phosphotungstic-acid 
hematoxylin. The treatment with permanganate of potas- 
sium and oxalic acid must be more prolonged than usual, 
however, otherwise the albuminous granules in the cytoplasm 
will stain too deeply and obscure the capillaries. 

For Eppinger's elaborate method, see Ziegler's Beitrage, 
vol. xxxi. 

Pancreas. — Much interesting and valuable work has been 
done recently on the histology of the pancreas, especially 
with reference to the cytoplasmic granules in the different 
kinds of cells of the ducts, glands, and islets. Some attempt 
has been made to apply the methods to the lesions of the pan- 
creas, more particularly to those associated with the syndrome 
known as diabetes mellitus. The results so far obtained are 
promising and encourage further study along the same lines. 

For routine microscopic study of the pancreas, fixation in 
Zenker's fluid and staining by the eosin-methylene-blue 
method are recommended. The zymogen granules do not 
stain intensely, as in the glands of the stomach and intestine 
after this procedure, but require special methods to render 
them prominent. Staining with phosphotungstic acid hema- 
toxylin after fixation in formaldehyde is sometimes useful. 
Fixation in formaldehyde is also useful for certain other 
purposes, such as the examination for fat, hemosiderin, and 
amyloid. 

As the best method for staining all the various granules in 
the cells of the pancreas, Bensley recommends that used for 
mitochondria (see page 107). 

For the specific granules of the A and B cells in the islets, 
the best technique in Bensley's opinion is the neutral gentian 
stain after fixation in chrome sublimate. 1 

1 For other methods of fixing and staining the islet cells consult these papers: 
Bensley, R. R., " Studies on the Pancreas of the Guinea Pig," Amer. Jour, of 
Anat, 297-388, xii., 191 1 ; Lane, M. A., "The Cytological Characters of the 
Areas of Langerhans," Amer. Jour, of Anat., vii., 1907. 



156 PATHOLOGICAL TECHNIQUE. 

A. Fix in the following solution: 

Potassium bichromate, 2.5 grams. 

Mercuric chlorid, 5.0 " 

Distilled water, 100.0 " 

It is Zenker's fluid minus the acetic acid, as the latter 
dissolves both mitochondria and the characteristic granules 
of the islet cells. 

The stain used is Bensley's neutral gentian, the name given 
to the neutral dye obtained when a solution of gentian violet 
is precipitated by its equivalent of a solution of Orange-G. 
The dye is prepared as follows : 

A. Gentian violet, 1 gram in 25 c.c. of water. 

B. Orange G., 1 gram in 25 c.c. of water. 

Add A to B, shaking gently, until practically complete 
precipitation has taken place. Filter and wash with water at 
once. Drain and dry. Dissolve residue in 25 c.c. of absolute 
alcohol. For staining, add the stock solution of the neutral 
compound to 20 per cent, alcohol until a solution having the 
color of a good hemalum solution is obtained. Allow this 
solution to stand twenty-four hours to permit the excess of 
dye to separate out, when it may be employed for staining as 
follows : 

1. Stain in neutral gentian violet solution twenty-four hours. 

2. Blot between several layers of filter paper. 

3. Dehydrate in acetone. 

4. Place sections in toluol. 

5. Differentiate in 

Absolute alcohol, I part ; 

Oil of cloves, 3 P arts 5 

6. Wash with toluol and mount in balsam. 

In the stain for mitochondria the granules in the A cells 
are stained deeply red, those in the B cells green. After the 
neutral gentian violet stain, followed by staining in acid 
fuchsin, the granules of the A cells are stained red, those of 
the B cells, violet. 

Goodpasture has found eosin and his acid polychrome 
methylene-blue solution to afford a very useful method for 



HISTOLOGICAL METHODS. 1 57 

staining differentially the zymogen and the alpha and beta 
granules after fixation in neutral Helly's fluid or in neutral 
Orth's fluid. 

1 . Fix thin pieces of fresh pancreas for twenty- four hours in 

Neutral formaldehyde, ioc.c. ; 

Zenker's fluid without acetic acid, 90 
Or in 

Neutral formaldehyde, 10 c.c; 

Bichromate of potassium (2.5 per cent.), 90 

2. Wash in running water twenty-four hours. 

3. Dehydrate in alcohol; embed in paraffin and cut sec- 
tions; pass through xylol and alcohol to water in the usual 
way. 

1. Potassium permanganate, 1 per cent, aqueous solution, 
one minute. 

2. Oxalic acid, 5 per cent, solution, one minute. 

3. Wash thoroughly in water. 

4. Stain in aqueous solution containing 1 per cent, eosin 
and 1 per cent, bichromate of potassium for one to five 
minutes. 

5. Wash hastily in water. 

6. Acid polychrome methylene-blue, one to five minutes. 

7. Wash hastily in water. 

8. Differentiate and dehydrate rapidly in 95 per cent, and 
absolute alcohol. 

9. Xylol and balsam. 

In properly stained sections zymogen granules stain deep 
purple; cytoplasm, light blue; nuclei, light purple; alpha 
granules, brick red, and beta granules, dark blue. 

Bone and Cartilage. — Excellent work can be done after 
hardening, in alcohol, and fixation in it is generally recom- 
mended for all infectious processes in bone. The histological 
structure is, however, better preserved in Zenker's or Orth's 
fluid. In decalcifying bone, after proper fixation, thin pieces 
should be taken, not more than 2 to 4 mm. thick, so that the 
process may be finished as quickly as possible. While tu- 
bercle bacilli will stain readily after being twenty-four or even 
forty-eight hours in 5 per cent, nitric acid, it is impossible 



158 PATHOLOGICAL TECHNIQUE. 

to stain them after they have been subjected to the same 
strength of nitric acid for four days. (For details in regard 
to decalcification see page 49.) 

Celloidin is preferable to paraffin for imbedding. Besides 
a simple stain with alum-hematoxylin, double stains of the 
latter with neutral carmine or eosin are sometimes advanta- 
geous. The best pictures with carmine as the contrast-stain 
are obtained by staining first in alum-hematoxylin, washing 
twelve to twenty-four hours, and then staining in the neutral 
carmine. The carmine stains decalcified bone and osteoid 
tissue red. Phosphotungstic-acid hematoxylin will some- 
times be found useful, especially when cartilage is present, 
because it stains the intercellular substance, both of bone and 
of cartilage, pink, while the nuclei are stained blue. The 
ground substance of cartilage, especially in new-growths, 
often stains so intensely with alum-hematoxylin that the 
nuclei are quite obscured. For the same reason chlorid of 
iron hematoxylin is often useful because it does not stain the 
ground substance. 

The following method is recommended for differentiating 
cartilage from bone : 

Schaffer's Safranin Method. — Decalcify with nitric acid. 

1. Stain sections a half to one hour in an aqueous solution 
of safranin, 1 : 2000. 

2. Wash in water. 

3. Place for two to three hours in a -^ per cent, solution 
of corrosive sublimate. 

4. Examine in glycerin, or, if permanent specimens are 
desired, pass very quickly through alcohol, blot with filter- 
paper, further dehydrate, and clear for a long time in berga- 
mot or clove oil, and mount in xylol balsam. This is a 
double stain : cartilage, orange ; bone, uncolored ; connective 
tissue and marrow, red. 

None of the methods above given has proved reliable in 
the study of rickets and of osteomalacia for differentiating 
osteoid from true bone-tissue. In important cases, therefore, 
it is advisable to use an old knife, and to cut sections of the 
undecalcified tissue after imbedding thoroughly in celloidin. 



HISTOLOGICAL METHODS. 1 59 

Schmorl's methods of demonstrating the lacunae and 
canalicular of bone in sections can be highly recommended. 

Method A. — 1. Fix, preferably in Muller's fluid, formal- 
dehyde, or Orth's fluid ; do not use corrosive sublimate 
solution. 

2. Decalcify by the slower methods — namely, Ebner's or 
Thoma's, or in Muller's fluids 100 c.c. plus nitric acid 3 c.c. 

3. Imbed in celloidin ; paraffin is objectionable. 

4. Place the sections for at least ten minutes in water to 
get rid of the alcohol. 

5. Stain for five to ten minutes or longer in saturated solu- 
tion of thionin in 50 per cent, alcohol, 2 c.c, water, 10 c.c, 
or in Nicolle's carbolthionin solution. 

6. Wash in water. 

7. Place in a saturated aqueous solution of picric acid for 
one-half to one minute. 

8. Wash in water. 

9. Place in 70 per cent, alcohol for about five to ten min- 
utes until no more dense clouds of color are given off. 

10. Dehydrate in 95 per cent, alcohol. 

11. Clear in oleum origani cretici. 

12. Xylol balsam. 

Bone substance yellow to yellowish-brown ; bone lacunae 
and canalicular dark brown to black; cells red. Fat-cells 
after fixation in Muller's fluid reddish violet. Osseous tissue 
stains a deeper yellow than osteoid tissue. Canalicular stain 
in osseous tissue, but not in osteoid tissue unless the thionin 
solution is made alkaline by the addition of I or 2 drops of 
ammonia. (This solution cannot be recommended for gen- 
eral use.) 

This method is not a true stain, but resembles Golgi's 
method ; a precipitation of coloring-matter takes place in 
the lacunae and canalicular ; it also takes place to a consid- 
erable extent in other narrow spaces in the tissues, and often 
is very disturbing. It can be gotten rid of to some extent 
without injury to the stain by leaving the sections in step 8 
in the water for half an hour. The canalicular are now 
usually brownish red to red, and the bone substance blue 



l60 PATHOLOGICAL TECHNIQUE. 

to colorless. In this case it is often best to stain the sec- 
tions first in alum hematoxylin to bring out the nuclei. 

Method B gives good results with the bones of children 
only. I. Harden in Mtiller's fluid or in Orth's fluid, fol- 
lowed by Miiller's for six to eight weeks, or for three to four 
weeks in the thermostat ; take very thin pieces of tissue. 

2. Wash off in water, and decalcify in Ebner's solution. 

3. Wash thoroughly in running water. 

4. Harden in alcohol ; imbed in celloidin ; cut sections 
very thin. 

5. Stain in Nicolle's carbolthionin, or better in the alkaline 
(NH 4 OH) thionin solution given above, for three minutes. 

6. Transfer to a saturated aqueous solution of phospho- 
tungstic or phosphomolybdic acid (use glass or platinum 
needle) for a few seconds or longer. The sections, become 
blue, green, or gray in color. 

7. Water five to ten minutes until they acquire a sky-blue 
color. 

8. Place in dilute ammonia (1-10) for three to five minutes 
to fix the color. 

9. Transfer directly to 90 per cent, alcohol ; change sev- 
eral times to get rid of the ammonia. 

10. 96 per cent, alcohol. 

11. Clear in carbol xylol. 

12. Xylol balsam. 

If the ground-substance is stained too deeply by the alka- 
line thionin solution, treat the sections with acid alcohol for 
five minutes, followed by water before dehydrating. The 
borders of the lacunae and canalicular stain bluish black; 
the ground-substance of bone clear to greenish blue ; cellu- 
lar elements a diffuse blue color. In rachitic bones the 
canalicular are brought out only in osseous tissue. 

Skin. — Much of the material for the study of lesions of 
the skin is obtained during life by means of the knife or 
scissors. Fixation in absolute alcohol is often advisable, es- 
pecially when it is desired to stain bacteria, mastzellen, plasma- 
cells, and elastic fibers. The staining methods for these 
tissue elements will be found on pages 107-119. For Unna's 



HISTOLOGICAL METHODS. l6l 

innumerable stains for degenerated connective-tissue fibers, 
elastic fibers, etc., the reader is referred to his numerous arti- 
cles on technique in the Monatsheft f. prakt. Dermatologie. 

For many skin-lesions, especially those in which blood- 
vessels play a more or less prominent part, Zenker's fluid is 
advisable. Clear tissues in oil of cedar wood rather than in 
chloroform for paraffin imbedding as it does not render them 
quite so brittle as chloroform does. 

In the examination of hairs or scales of epidermis for 
bacteria and fungi it is important first to remove the fat from 
them by means of equal parts of alcohol and ether. They 
are then examined in 40 per cent, caustic potash, which, by 
clearing up the cells, brings out the organisms and spores 
quite distinctly. Heating the potash over a small flame 
hastens the process, but is a somewhat risky proceeding ; 
soaking in the solution over night is better. Examine the 
preparation with most of the light excluded. 

Preparations may be made in certain cases by touching the 
cover-slip to the surface of the lesion, drying, and passing 
through the flame. After removing the fat by means of alco- 
hol and ether, stain as with ordinary cover-slip preparations. 

Unna's method is to rub up the scales of epidermis in a 
little glacial acetic acid between two slides, which are then 
drawn apart and quickly dried over the flame. After re- 
moving the fat by means of alcohol and ether the slide 
preparations are stained in borax-methylene-blue. 

For staining the various vegetable parasites of the skin 
Malcolm Morris recommends the following method, which 
he claims is the best orte yet devised, as it avoids the use of 
the hydrate of potash : 

1. Ether or alcohol and ether equal parts. 

2. Stain in a solution of 5 per cent, gentian-violet in 70 
per cent, alcohol, five to thirty minutes. 

3. Iodin solution, one minute. 

4. Aniline, or aniline plus 2 to 4 drops of nitric acid. 

5. Aniline. 

6. Xylol. 

7. Xylol balsam. 

11 



1 62 PATHOLOGICAL TECHNIQUE. 

The most suitable medium for the growth of the various 
ringworms is the following, due to Sabouraud : 

Agar-agar, 1.30; 

Peptones, .50; 

Maltose 3.80 ; 

Water, 100. 

Instead of test-tubes, Erlenmeyer flasks are used, so as 
to get a large flat surface for the growth to spread over from 
the point of inoculation in the center. The most favorable 
temperature for growth is 30 C. 

Museum Preparations. — Specimens intended to be 
preserved for the museum should generally be gotten into 
pretty good shape by trimming and dissecting before they 
are placed in the hardening reagent. Of the liver or other 
large organs and tumors, sections several centimeters thick 
are generally preferable to the whole specimen. The usual 
custom in the past has been to wash the specimen for a 
number of hours or over night in running water, to get rid 
of the blood, and then to preserve in 80 per cent, alcohol. 
This method preserves form and relations well, but is nearly 
valueless for preserving colors. 

Since the introduction of formaldehyde, from which at 
first much was expected in the way of faithful fixation of 
the normal colors of gross preparations, numerous attempts 
have been made to improve on the results obtainable with 
formaldehyde alone. Of the methods advocated, the follow- 
ing from Virchow's laboratory seems the most promising, 
and can be highly recommended : 

Kaiser-ling's Method of Preserving 1 the Natural Colors 
in Museum Preparations. — 1. Fixation for one to five days 
in — 

Formaldehyde, 200 c.c. ; 

Water, 1 000 " 

Nitrate of potassium, 1 5 grams ; 

Acetate of potassium, 30 " 



HISTOLOGICAL METHODS. 163 

Change the position of the specimen frequently, using rubbei 
gloves to protect the hands from the injurious effect of the 
formaldehyde. The time of fixation varies with the tissue 
or organ and size of the specimen. 

2. Drain and place in 80 per cent, alcohol one to six 
hours, and then in 95 per cent, alcohol for one to two hours, 
to restore the color, which is somewhat affected in the 
fixing solution. 

3. Preserve in — 



Acetate of potassium, 


200 grams ; 


Glycerin, 


400 c.c. 


Water, 


2000 " 



Exposure to light gradually affects the colors. The 
process of fixation, should be performed in the dark, and the 
specimens when preserved should be kept in the dark except 
when on exhibition. 

If it seems desirable to cut a thin slice from the face of a 
specimen, this should not be done until the preparation has 
been in the preservative fluid two weeks. The specimen 
may then be placed in alcohol for one to two hours to 
brighten up the colors. 

It is advisable to add camphor, thymol, carbolic acid (one 
per cent.), or some other preservative to the third solution 
to prevent the growth of molds. 

Pick has modified the steps of Kaiserling's method as 
follows : 

1. Fixation up to five days in — 

Water, 

Formaldehyde, 
Carlsbad salt, 

2. Eighty to 85 per cent, alcohol. 

3. Water, 
Glycerine, 
Acetate of sodium. 



OOO c.c. ; 


50 c.c. ; 


50 gm. 


900 c.c. ; 


540 c.c. ; 


270 gm. 



164 PATHOLOGICAL TECHNIQUE. 

For display purposes the preparations preserved by these 
methods are often mounted permanently in gelatin. A jelly 
is made by adding ten parts by weight of gelatin to the 
third solution in which the specimens are ordinarily kept. 
The same procedure should be followed as in the making 
of Kaiser's glycerine jelly for the mounting of histological 
sections. For a preservative add one per cent, of carbolic 
acid. Formaldehyde is often used for this purpose, but in 
spite of it, or owing to its evaporation, ferments present in 
the tissues (as shown by L. J. Rhea) often result in liquefac- 
tion of the jelly in the course of weeks to months. 

Owing to the discoloration which sometimes takes place 
in the jelly and the difficulty of remounting the tissues, 
L. W. Williams advises using only a layer of the jelly suf- 
ficient to attach the specimen to the back of the jar, and 
filling up the space in front with the third solution, which 
can easily be renewed. By this method the specimen is held 
firmly in place and is viewed through a clear, colorless 
medium. 



PATHOLOGICAL PRODUCTS. 

Cloudy Swelling; Albuminous Degeneration. — 

The increase in the relative number of the albuminous 
granules of the cytoplasm of the various tissue-cells in 
pathological processes is usually determined by examination 
of the fresh material, either macroscopically from the appear- 
ances on section, or microscopically from teased preparations 
or frozen sections mounted in salt solution. The organ as a 
whole (and therefore the individual cells) usually shows 
some increase in size. The nucleus is generally more or 
less obscured if the process is at all marked. According to 
Israel, the cloudiness must be recognizable with low powers 
and in places where the cells are massed together. The 
diagnosis should not be based on the appearances of single 
cells. 

The chemical properties of the albuminous granules are 
the following : they disappear on treatment with dilute acetic 



HISTOLOGICAL METHODS. 



65 



acid (1-2 per cent, solution usually) ; they are not dissolved 
by chemical substances which dissolve fat (absolute alcohol, 
ether, chloroform, etc.) ; and they do not stain with osmic 
acid. The acetic-acid test is the one usually employed. 

Albuminous degeneration can also be studied in sections 
of tissues hardened in certain of the fixatives and stained 
with diffuse colors. For this purpose hardening in Zenker's 
fluid and staining in eosin and methylene-blue or in alum- 
hematoxylin and eosin can be highly recommended. 

Fat. — This term includes a variety of fat-like substances, 
of which the three most common and important in the 
animal kingdom are olein, palmitin, and stearin, the glycerin- 
ates of oleic, palmitic, and stearic acids. Under certain con- 
ditions the free fatty acids themselves and their lime-salts 
may be present. Among the fat-like substances belongs 
also the myelin of the nerve-sheaths. The pigments of the 
ganglion-cells and of many other cells contain fat, and hence 
react like fat, but the fat and the pigment can be separated. 

These various forms of fat differ somewhat in their reac- 
tion to certain reagents used as tests. They are all insoluble 
in dilute acids and alkalies, but are soluble in strong alcohol, 
in ether, and in chloroform. Osmium tetraoxid is reduced 
by olein and oleic acid, by myelin, and the fat in certain pig- 
ments, but is not reduced by the palmitic and stearic com- 
ponents of body-fat unless the tissue after exposure to the 
osmium tetraoxid is placed in dilute alcohol ; if placed in 
strong alcohol, the reduction takes place imperfectly or not 
at all. Myelin differs from the other fat substances in that 
it can be fixed by a chrome salt so that it will no longer 
reduce the osmium tetraoxide. 

Scharlach R. is the strongest of a group of fat stains of 
which Sudan III. has, perhaps, been the most used until 
recently. These stains have the common property of dis- 
solving readily in all fats, including myelin and the lipo- 
chrome of ganglion-cells. The staining, which is purely 
physical in nature, depends simply on the fact that fat is a 
better solvent of the stain than the alcohol is, and takes it 
up from it. 



1 66 PATHOLOGICAL TECHNIQUE. 

The common tests for fat are the three following: As all 
these fatty substances are unaffected (at least not for some 
months) by formaldehyde, it is becoming more and more 
customary to apply the tests, especially the Scharlach R. 
stain, to frozen sections after fixation in that reagent 

1. Acetic Acid. — The test may be applied to teased 
preparations or to frozen sections. The acetic acid is gener- 
ally employed in a I or 2 per cent, solution, of which a few 
drops are placed at the edge of the cover-slip and drawn 
under by means of a bit of filter-paper placed at the oppo- 
site edge. Albuminous granules disappear optically while 
fat-droplets persist. 

2. The Osmium Stain for Pat. — A I per cent, solution 
may be applied like the acetic-acid test to teased prepara- 
tions or to frozen sections. It is more usual, however, to 
fix very thin sections (not over I mm. thick) in a I per cent, 
solution of osmium tetraoxid for one to three days, or, 
better still, in a combined solution, such as Flemming's or 
Marchi's, containing it. 

Flemming's solution should be allowed to act from two to 
four days if the tissue is from 2-3 mm. thick, and then the 
pieces of tissue should be thoroughly washed in running 
water for twenty-four hours before being placed for several 
days in dilute and then in strong alcohol. 

Marchi's method was intended for differentiating fat from 
myelin (see page 48), but the solution employed by him 
may be used for staining fat in ordinary tissues. Place small 
pieces of tissue in it for five to eight days, wash thoroughly 
in running water, and place in dilute (50 to 70 per cent.) 
alcohol for several days before transferring to strong alcohol. 

Marchi's method, carried through in the manner just de- 
scribed, succeeds perfectly with tissues fixed in formaldehyde. 

Osmium reduced by fat is soluble in ether, turpentine, 
xylol, and toluol, but is not dissolved by alcohol, chloro- 
form, or oil of cloves. Steensland adds oleum origani cretici 
to this list and recommends it for clearing celloidin sections. 
Imbedding in celloidin is not contra-indicated, as the alcohol 
probably protects the osmium from the injurious action of 



HISTOLOGICAL METHODS. 1 67 

the ether. For the paraffin method clear in chloroform, and 
mount in properly prepared chloroform balsam (see page 
106). 

3. The Scharlach R. Stain for Pat. — The solution of 
Scharlach R. most commonly used has been a saturated one 
in 70 per cent, alcohol. It requires staining overnight in 
order to obtain the best results. Recently, G. Herxheimer 
has advised a saturated solution in — 

Seventy per cent, alcohol, 50 c.c. ; 

Pure acetone, 50 " 

Staining takes place very quickly and is intense and sharp. 
The solution can be highly recommended and should take 
the place of any other yet proposed. 

Cover-slip Preparations. — 1. Fix in the vapor of formalde- 
hyde for five to ten minutes. 

2. Stain in the alcohol-acetone solution of Scharlach R. 
for two to five minutes. 

3. Dip for an instant in 70 per cent, alcohol. 

4. Wash in water. 

5. Counterstain with alum hematoxylin or methylene-blue. 

6. Wash in water. 

7. Mount in glycerin or glycerin jelly. 

Sections. — 1. Make frozen sections of formaldehyde-fixed 
tissue. 

2. Dip for an instant in 70 per cent, alcohol. 

3. Stain in the alcohol-acetone solution of Scharlach R. 
for two to five minutes. 

4. Wash quickly in 70 per cent, alcohol. 

5. Counterstain in alum hematoxylin. 

6. Wash thoroughly in water. 

7. Mount in glycerin or glycerin jelly. 

The staining should always be done in a tightly stoppered 
bottle, because with any evaporation of the alcohol a precipi- 
tation of the staining material immediately takes place. 

If, after staining with alum-hematoxylin, the sections are 
put into a 1 per cent, aqueous solution of acetic acid for three 



1 68 PATHOLOGICAL TECHNIQUE. 

to five minutes, the color of the nuclei is a clearer blue, in 
better contrast with the red color of the fat, and the stain- 
ing is sharper. 

Kaiser's Glycerin Jelly — 

Finest French gelatin, 40 grams; 

Water, 210 c.c; 

Glycerin, 250 
Carbolic acid crystals, 5 grams. 

Soak the gelatin in the water for two hours. Add the 
glycerin and the carbolic acid and warm for two to fifteen 
minutes, stirring all the while until the mixture is smooth. 
It is advised to filter through the finest spun glass laid wet 
in a funnel. The solution will, however, filter through filter- 
paper in the course of twenty-four hours if placed in the 
paraffin oven (temperature of about 54 C). Glycerin jelly 
is much to be preferred to glycerin because the mounts are 
practically permanent; the cover-slips are fixed. 

Benda's Stain for Fat Acid Crystals. — In a solution of 
copper acetate the crystals of the free fatty acids and of the 
fatty acid salts ot lime-stain blue. Benda recommends fixa- 
tion in Weigert's copper-fluorchrom-acetic-acid-mordant plus 
10 parts of formaldehyde for two to four days at 37 C. 
Frozen sections are then to be cut, counterstained in Schar- 
lach R. and alum-hematoxylin, and mounted in glycerin. 

This method has been modified by Fisher, and later by 
Klotz, as follows : 

1. Fix the tissue and precipitate the fatty-acid radical in 
the following solution for one to twenty-four hours : 

Chromalum, 2.5 grams. 

Formaldehyde, 4 per cent., 100 c.c. 

Dissolve by boiling ; while cooling add — 

Glacial acetic acid, 5 c.c. 

And then powdered neutral acetate 

of copper, 5 grams. 

2. Wash thoroughly in water. 



HISTOLOGICAL METHODS. 1 69 

3. Cut sections on freezing microtome, 

4. Stain sections in a saturated solution of hematoxylin in 
60 per cent, alcohol for six hours. 

5. Wash in water and treat with the following fluid (Wei- 
gert's decolorizing fluid) until the tissue becomes a light 
brown, while the sites of the fatty-acid radical remain black : 

Potassium ferricyanide, 2.5 grams. 

Borax, 2.0 " 

Water, distilled, 100.0 c.c. 

6. Water, alcohol, balsam. 

After washing in water the sections may be stained with 
Scharlach R. and mounted in glycerin. 

Cholesterin crystals are recognized by their shape. On the 
addition of concentrated sulphuric acid the crystals turn 
yellow and then rose-color. Treated with a little iodin, fol- 
lowed by concentrated sulphuric acid, they become colored 
violet, changing gradually to blue, green, and red. 

Necrosis. — Necrosis in tissues is generally recognized by 
two features : either by the disappearance of the nuclei, al- 
though the cell-outlines may be visible, so that the nuclear 
stain is no longer possible, or by the presence of irregular, 
larger or smaller masses, generally supposed to be due to a 
fragmentation or breaking-up of the chromatin, which stain 
intensely with nuclear stains. The disappearance of the 
nucleus is not synchronous with the death of the cell, but 
begins some twenty-four hours later, so that it is really evi- 
dence of changes following necrosis. It follows from the 
above that the microscopic evidence of necrosis is best 
studied in sections of tissues hardened in fixatives which 
favor nuclear staining, such as Zenker's fluid, formaldehyde, 
etc. Teased preparations and frozen sections of fresh tissue 
are much less useful. 

For the study of sections from hardened material double 
stains with alum-hematoxylin and eosin. or, still better, with 
eosin followed by Unna's alkaline methylene-blue solution, 
after Zenker's fixation, are very useful, for the reason that 
the necrotic areas usually stain rather deeply with the diffuse 



170 PATHOLOGICAL TECHNIQUE. 

stain, and are thereby brought out sharply. This is particu- 
larly true of necroses of the liver. 

For rendering the fragmented nuclei prominent the same 
methods may be followed as for mitosis. A fuchsin stain 
washed out by picric acid in the alcohol will often give 
excellent results. 

Caseation is probably a tissue-change following local 
necrosis. Macroscopically and microscopically it resembles 
harder or softer cheese. Under the microscope it appears 
as coarsely or finely granular masses which have more or 
less completely lost the original tissue-structure. The chemi- 
cal changes which have taken place have not been studied. 
Fibrin may or may not be present. Caseous tissue possesses 
no peculiar staining reactions. Fragmented nuclei are fre- 
quently present in it, especially in the peripheries of the 
areas. 

Fibrin. — Fibrin usually appears as delicate, transparent, 
slightly refractive threads which are often closely matted 
together so as to form large masses. More rarely it appears 
as coherent masses of the finest granules, as homogeneous 
glassy lumps, or as thin sheets. The characteristic reaction 
for fresh fibrin is that it quickly swells up and optically dis- 
solves in very dilute acetic acid. 

Fibrin is well brought out in sections of hardened tissues 
by a double stain of alum-hematoxylin and eosin, or of eosin 
followed by Unna's alkaline methylene-blue solution, espe- 
cially if the specimens have been fixed in Zenker's fluid. 
Two other stains which bring it out with great sharpness 
are phosphotungstic-acid-hematoxylin and the aniline-blue 
method for collagen fibrils. 

Weigert's Differential Stain for Fibrin. — i. Harden in 
alcohol. 

2. Stain sections in lithium carmine (see page 92). 

3. Stain in Weigert's aniline-methyl-violet three seconds. 

4. Wash off with normal salt solution. 

5. Weigert's iodin solution a few seconds. 

6. Wash off with water. 

7. Decolorize in aniline oil and xylol equal parts. 



HISTOLOGICAL METHODS. I7 1 

8. Wash off with three changes of xylol. 

9. Xylol balsam. 

To stain sections fixed in formaldehyde or in a chrome 
salt, place them in a \ per cent, aqueous solution of per- 
manganate of potassium for ten minutes, wash in water, and 
reduce in a 5 per cent, aqueous solution cf oxalic acid for 
two to three hours or longer. Then wash thoroughly in 
water. 

The fibrin and those bacteria which are stained by Gram 
are stained blue. The nuclei are red if the decoiorization is 
carried far enough. It can easily be watched under the low 
power of the microscope. The method is not always suc- 
cessful, especially with tissues which are old. Besides the 
fibrin, certain forms of hyalin are often stained by this 
method. 

Differential stains for fibrin are also obtained by the chlo- 
rid of iron hematoxylin stain (page 90) and by the con- 
nective-tissue stain (page 11 1). The former is applicable 
after any fixing reagent, the latter only after Zenker's 
fluid. 

The phosphotungstic-acid-hematoxylin method is also 
useful if all the steps are followed out as for neuroglia fibrils. 

Mucin.— The term " mucin " is applied to a proteid sub- 
stance having certain chemical reactions, and also to certain 
other substances which give the same reactions, but do not 
belong to the proteids. These various substances of secre- 
tory and degenerative origin cannot be distinguished micro- 
scopically, and have been investigated but little chemically. 
The reactions in common are the following : they dissolve 
in water to form a slimy fluid ; they are precipitated from 
slightly alkaline solutions by acetic acid ; the fresh precipi- 
tate dissolves in alkalies and in neutral salt solutions. Acetic 
acid, usually employed for this purpose in a 1 or 2 per cent, 
solution, precipitates mucin in the form of threads or gran- 
ules. This reaction with fresh tissues has long been the 
main test for mucin. The acetic acid is drawn under the 
cover-slip by means of filter-paper placed at the opposite 
edge. The preparation should be mounted in water, not in 



172 PATHOLOGICAL TECHNIQUE. 

salt solution, which may hinder or entirely prevent the reac- 
tion from taking place. Of late certain color reactions have 
become prominent. Mucin is coagulated into threads by 
alcohol or corrosive sublimate, and in this form can be 
stained by a number of staining reagents. Alum-hema- 
toxylin under certain conditions will stain mucin. Accord- 
ing to P. Mayer, these conditions depend on a certain degree 
of ripeness of the solution, on the presence of enough alum 
to keep the nuclei from staining deeply, and, most important 
of all, on the absence of any free acid. This is difficult to 
manage, unless the solution is carefully neutralized, on ac- 
count of the acid properties of alum. Mayer, therefore, 
recommends staining the sections in muchematein. 



Mayer's Muchematein. — 




Hematein, 


0.2 grams ; 


Chlorid of aluminum, 


0.1 " 


Glycerin, 


40 c.c. ; 


Water, 


60 " 



Rub up the hematein with a few drops of glycerin and the' 
chlorid of aluminum, and dissolve the mixture in the glycerin 
and water. Mucin appears blue : the other tissue elements 
are not stained. 

Various aniline dyes have been recommended for staining 
mucin : those most favorably spoken of are methylene-blue 
(Orth), Bismarck brown (P. Mayer), thionin (Hoyer), poly- 
chrome methylene-blue (Unna), and toluidin-blue. The 
drawback to most of the aniline stains is that they are 
quickly extracted by the alcohol used for dehydrating. On 
this account P. Mayer highly recommends Bismarck brown, 
because permanent mounts can be easily made with it. It 
is not extracted by alcohol, and it does not fade in Canada 
balsam like many of the others. 

Hardening in corrosive sublimate and imbedding in par- 
affin are generally recommended as preferable to hardening 
in alcohol and imbedding in celloidin. Stain sections for 
five to fifteen minutes in a rather dilute aqueous solution of 
the dve chosen. Of Bismarck brown use a saturated aque- 



HISTOLOGICAL METHODS. 1 73 

ous solution, and stain, if necessary, twenty-four hours. 
With thionin, toluidin-blue, and polychrome methylene-blue 
metachromatic stains are obtained ; the mucin is colored red, 
the rest of the tissue blue. Two special methods for stain- 
ing mucin are given in detail : 

Hoyer's Thionin Stain. — Mucin, red ; everything else, 
blue. I. Harden in corrosive sublimate, followed by alcohol. 

2. Paraffin sections are passed through xylol, chloroform, 
and 95 per cent, alcohol to free them from paraffin, and are 
then placed in a 5 per cent, aqueous solution of corrosive 
sublimate for three to five minutes. 

3. Stain in a dilute solution of thionin for ten to fifteen 
minutes. 

4. Alcohol. 

5. Clear in the mixture of the oils of cloves and thyme. 

6. Turpentine oil or oil of cedar. 

7. Balsam. 

Before the staining the sections must not be treated with 
iodin solution to get rid of the precipitate of mercury, be- 
cause it spoils the staining. 

Unna's Polychrome Methylene-blue Stain. — 1. Fix in 
alcohol. Stain paraffin or celloidin sections in polychrome 
methylene-blue five to ten minutes or longer. 

2. Wash in acidulated water. 

3. Fix in 10 per cent, solution of bichromate of potassium 
half a minute. 

4. Wash in water. 

5. Dry on slide with filter-paper. 

6. Decolorize in aniline plus 1 per cent, hydrochloric acid 
(a few seconds only). 

7. Wash off with oil of bergamot. 

8. Balsam. 

Nuclei blue, mucin, cartilage, and amyloid red. 

Pseudo-mucin dissolves in water to form a slimy 
material, and is precipitated from its solutions by alcohol in 
thread-like masses which are again soluble in water. It is 
not affected by acetic acid. Pseudo-mucin is found in cer- 
tain ovarian and other tumors. 



174 PATHOLOGICAL TECHNIQUE. 

Colloid and Hyalin. — The terms colloid and hyalin 
are not yet sharply limited to definite chemical substances. 
The term colloid was originally applied to the homogeneous 
substance found in the thyroid gland, but has been broad- 
ened to include various substances of a similar appearance. 
The term hyalin is still more indefinite, but its use may be 
said to be applied most generally to those homogeneous 
substances which stain deeply with various stains, in contra- 
distinction to those which, like colloid, show no marked 
affinity for staining reagents after ordinary fixatives. 

Unquestionably, numerous substances of different chemi- 
cal composition and of varying origin have been grouped 
under these two titles because of their physical and optical 
characteristics — namely, that they occur as glassy, refractive, 
homogeneous, occasionally colored gelatinous or firm masses. 
Chemically, very little that is definite is known about them, 
and they possess no peculiar chemical reactions. Several at- 
tempts have been made to classify them in accordance with 
their reactions to various staining reagents. 

Von Recklinghausen applied the term colloid to all the 
homogeneous, transparent-looking substances, including mu- 
cin, amyloid, etc., and reserved the term hyalin for a special 
group, which, according to him, is characterized by the fol- 
lowing peculiarities : it resembles amyloid in physical charac- 
teristics, but does not react to iodin ; it stains deeply with 
acid dyes, such as eosin and acid fuchsin. 

Ernst has recently endeavored to differentiate two groups 
of hyaline substances, colloid and hyalin, by means of their 
reaction to Van Gieson's picro-acid fuchsin solution. Ac- 
cording to him, true hyalin stains with acid fuchsin alone, 
and appears of a deep-red color, while colloid, of which the 
typical example is found in the thyroid gland, stains with 
both picric acid and acid fuchsin, so that it appears of an 
orange or yellowish-brown color. He has also tried to 
prove that all colloid is derived from epithelial cells, while 
all hyalin comes from connective tissue or from blood-vessels. 

According to Von Kahlden, these differential staining re- 
actions with Van Gieson's mixture claimed by Ernst for col- 



HISTOLOGICAL METHODS. 



175 



bid and hyalin are by no means justifiable, because true 
colloid often stains a deep red. Furthermore, Unna has 
shown that in the skin connective-tissue cells can give rise to 
the so-called true hyalin, of which part is acidophilic and 
part basophilic, while the intercellular substance gives rise to 
colloid. 

The last attempt to classify the various homogeneous sub- 
stances on the basis of their reactions to dyes, apparently 
the only method possible at present, has been made by 
Pianese as a result of his studies of the various degenerative 
processes occurring in cancer-cells. He used a special fixa- 
tive (see p. 49) and five different staining methods (see p. 
80, methods III. A. and B., IV., V., and VI.). Of these 
methods, III. B. is the best, because it gives a characteristic 
color to each substance — hyalin, brick-red; colloid, bright 
green ; mucin, clear sky-blue ; and a substance resembling 
amyloid, a dark reddish-violet. Besides these distinct re- 
actions for colloid, hyalin, mucin, and a substance resem- 
bling amyloid, he found others less definite ; one of these he 
calls pseudo-mucin and another pseudo-colloid. As a basis 
for his studies he took the reactions of amyloid, mucin (in- 
testine), colloid (thyroid gland), and hyalin (hyalin remains 
of ovarian follicles, hyaline degeneration of renal glomeruli), 
with the same stains after fixation in his own hardening 
mixture. 

The above brief historical statement is considered neces- 
sary to show the present views in regard to these various, 
more or less indefinite, homogeneous, transparent substances. 
For demonstrating them after the usual hardening reagents, 
of which alcohol and corrosive sublimate are perhaps the 
best, a double stain with alum-hematoxylin and eosin is very 
useful. Certain of the homogeneous substances stain deeply 
with eosin ; others, like the transparent drops and masses 
occasionally found in the walls of the blood-vessels of the 
brain, stain with hematoxylin. Sometimes good results can 
be obtained with Weigert's fibrin stain or with carbol-fuch- 
sin. The most generally useful stain, aside from alum-hema- 
toxylin and eosin, is probably Van Gieson's mixture. 

The hyalin in liver-cells in alcoholic cirrhosis stains deeply 



I?6 PATHOLOGICAL TECHNIQUE. 

by the eosin-methylene-blue method after fixation in Zenker's 
fluid. The color varies from blue to red, depending, to some 
extent at least, on the freshness of the tissue. It stains deep 
blue in phosphotungstic-acid-hematoxylin preparations. 

1. Stain deeply in alum-hematoxylin. 

2. Wash in water. 

3. Stain three to five minutes in a saturated aqueous solu- 
tion of picric acid, to which is added enough of a saturated 
aqueous solution of acid fuchsin to give it a deep-red color. 
The effect of various proportions is sometimes useful. 

4. Wash in water. 

5. Alcohol. 

6. Oleum origani cretici. 

7. Balsam. 

The transparent homogeneous substances usually stain 
from orange to deep red in color ; connective tissue, red. 

Unna's Method for Hyaline and Colloid Material. — A. 
Harden in alcohol. 1. Acid fuchsin (2 per cent, aqueous 
solution) five minutes. 

2. Saturated aqueous solution of picric acid two minutes. 

3. Saturated alcoholic solution of picric acid two minutes. 

4. Wash off in alcohol. 

5. Oil, balsam. 

Hyaline and connective-tissue fibers, red ; colloid of thy- 
roid gland, yellow ; protoplasm, yellow. 

B. To show acidophilic and basophilic hyaline : 1. Water- 
blue (2 per cent, aqueous solution), twenty to thirty seconds. 

2. Water. 

3. Carbol-fuchsin one to two minutes. 

4. Water. 

5. Alcohol slightly tinged with iodin. 

6. Pure alcohol. 

7. Oil, balsam. 

Nuclei, keratin, and large hyaline masses, cherry red ; con- 
nective-tissue fibrillar, protoplasm, and small hyaline bodies, 
blue. 

For finer work the methods of Pianese should be used. 

Keratohyalin (Unna). — I. Stain sections in a fairly old 
alum-hematoxylin solution until they are over-stained. 



HISTOLOGICAL METHODS. 1 77 

2. Place in a very weak solution of permanganate of 
potassium (about I : 2000) for ten seconds. 

3. Dehydrate and decolorize in alcohol. 

4. Oil, balsam. 

An isolated stain of the granules of keratohyalin is ob- 
tained, blue-black in color. 

In like manner a 33 per cent, solution of sulphate of iron 
acting for ten minutes, or a 10 per cent, solution of chlorid 
of iron for a few seconds, will produce the same effect. Or- 
dinarily, sections are stained deeply in alum-hematoxylin, 
and decolorized with acetic acid and alcohol or with hydro- 
chloric acid and alcohol. 

Glycogen. — Glycogen is a carbohydrate of slightly vary- 
ing composition, occurring in cells and nuclei, more rarely in 
the intercellular tissue, either diffusely or more commonly in 
the form of larger and smaller masses and granules of a trans- 
parent homogeneous appearance. It is demonstrated micro- 
chemically by means of its reaction with iodin, which stains 
it brown. It is easily differentiated from amyloid by the 
fact that with the exception of the glycogen from certain 
sources, such as cartilage-cells, it is readily soluble in water 
and does not give the iodin-sulphuric-acid reaction. 

In consequence of its property of dissolving readily in 
water the aqueous Lugol's solution of iodin cannot be em- 
ployed for staining glycogen in fresh tissues. Instead, a thick 
solution of gum arabic containing 1 per cent, of Lugol's 
solution must be used, or, better still, equal parts of glycerin 
and Lugol's solution, in which the sections are more perfectly 
cleared. 

For sections hardened in absolute alcohol the same 
methods may be used, but better results, and practically 
permanent mounts, can be obtained by the method of Lang- 
hans. Lugol's solution is used for staining the sections, be- 
cause after hardening in alcohol the glycogen is much less 
soluble in water than in the fresh state. The iodin-glycerin 
mixture would probably be better. Best's carmine stain, how- 
ever, affords, by all odds, the most brilliant permanent and 
satisfactory method of demonstrating glycogen in the tissues, 

and has practically superseded the use of iodine in any way. 
12 



178 PATHOLOGICAL TECHNIQUE. 

I. Lang-hans' Iodin Stain. 

1. Stain paraffin sections in Lugol's solution. 

2. Dehydrate in 1 part of tincture of iodin to 3 or 4 parts 
of absolute alcohol. 

3. Clear in oleum origani cretici. 

The sections are to be preserved in oil. Even a ring of 
balsam around the cover-slip will cause the color to fade. 
Other oils are not so good. 

2. Lubarsch's Iodin- Hematoxylin Stain. 

1. Fixation in absolute alcohol. 

2. Stain paraffin sections for five minutes in the following 
solution, which should be filtered and carefully protected 
from sunlight : 

Delafield's hematoxylin, 2 ; 

Lugol's solution, 2 ; 

Water, I. 

3. Absolute alcohol, xylol, xylol balsam. 

4. Expose to daylight one to two days. Glycogen brown, 
nuclei blue. 

Lubarsch's Gentian-violet Stain. — Alcohol fixation ; 
paraffin imbedding. 

1. Stain with Meyer's alcoholic carmine solution, differen- 
tiate in acid alcohol, wash off in absolute alcohol. 

2. Stain in aniline oil gentian-violet for one to two minutes, 
warming slightly if necessary. 

3. Wash quickly in water. 

4. Gram's solution of iodin on section continuously for 
five to ten seconds. 

5. Dry thoroughly with filter-paper. 

6. Dehydrate and differentiate in aniline-oil xylol (2 to 1) 
or in pure aniline oil. 

7. Wash thoroughly in xylol and mount in balsam. 
Nuclei red ; glycogen dark blue to violet. It is advisable 

to expose the sections to daylight for one to two days. The 
preparations will keep up to one year. 

Best's Carmine Stain. — Fix tissues in alcohol : formalde- 
hyde and corrosive sublimate are not so good. 



HISTOLOGICAL METHODS. 1 79 

Imbed in celloidin, which prevents the glycogen from dis- 
solving in water. Paraffin and frozen sections should not 
be used. 

The stock carmine solution is made as follows : 

Carmine, 2.0 grams. 
Potassium carbonate, 1.0 " 

Potassium chlorid (KG), 5.0 " 

Aq. dest., 60.0 c.c. 

Boil gently and cautiously for several minutes. 
After cooling add — 

Liq. ammon. caustic, 20.0 c.c. 

In tightly stoppered bottles this solution will keep and be 
available for staining glycogen for two months in winter, and 
for about three weeks in summer. 

The staining method is as follows : 

1. Stain sections deeply with alum hematoxylin. 

2. Decolorize with acid alcohol, if necessary. 

3. Wash thoroughly in running water. 

4. Stain sections for five minutes in the following solution : 

The above carmine solution (freshly 

filtered), 2.0 c.c. 

Liq. ammon. caustic, 3.0 " 

Methyl alcohol, 3.0 " 

5. Differentiate in — 

Alcohol abs., 80.0 c.c. 

Methyl-alcohol, 40.0 " 

Aq. dest., 100.0 " 

from three to five minutes, changing the fluid occasionally 
until it remains uncolored. 

6. Wash off in 80 per cent, alcohol. 

7. Alcohol, oil, balsam. 

Glycogen red, nuclei blue. The method also stains the 
peptic cells of the stomach, the corpora amylacea of the 
nervous system, and sometimes the mucin in goblet-cells 
and the Granules of mast-cells. 



l8o PATHOLOGICAL TECHNIQUE. 

Caution : Do not put the sections into water after steps 
4 or 5, because the carmine will diffuse out of the specimens. 

Amyloid Infiltration. — Amyloid is a combination of an 
albuminous body with chondroitin sulphuric acid. It is 
insoluble in water, alcohol, ether, and dilute acids, and is 
not digested by pepsin and hydrochloric acid. It is distin- 
guished from the other homogeneous substances, except 
glycogen, by the fact that it is stained mahogany-brown by 
iodin in solution. The reaction is particularly useful as a 
test on fresh gross material. If a section containing amyloid 
be quickly and lightly stained in Gram's iodin solution and 
then transferred to sulphuric acid, the color of the amyloid 
will usually change at once or in a few minutes from red, 
through violet, to blue. Sometimes the color turns simply 
of a deeper brown. Several of the aniline dyes give almost 
as perfect characteristic color reactions for amyloid as iodin, 
and are perhaps better for the purposes of histological 
study. Any of these differential stains may be used with 
fresh or hardened tissues. Alcohol as a hardening reagent 
gives the best results, but the other fixatives may be em- 
ployed. Unfortunately, good permanent mounts cannot 
be made with any of the characteristic stains, so that the 
ordinary double stains of alum-hematoxylin with eosin or 
Van Gieson's mixture will often be found of the greatest help 
in studying the distribution of amyloid. The aniline-blue 
connective-tisue stain can also be highly recommended 
because it stains amyloid light blue, so that it stands out in 
marked contrast to the red of the liver-cells. 

Iodin Reaction for Amyloid. — I. Stain sections in a weak 
solution of iodin (Lugol's solution diluted until of a clear 
yellow color) for three minutes. 

2. Wash in water. 

3. Mount and examine in water or glycerin. 

If the tissue reacts strongly alkaline, a condition which 
may result from post-mortem decomposition, the color reac- 
tion with iodin will not take place. In such cases the tissue 
or the sections of it should be treated with dilute acetic acid 
before applying the test. The normal reaction of amyloid 



HISTOLOGICAL METHODS. 



181 



with iodin may be increased by treating the section after 
staining with dilute acetic acid. 

Langhans' Method for Obtaining* Permanent Mounts 
with Iodin. — I. Harden in alcohol and stain in Mayer's alco- 
holic carmine solution. 

2. Stain sections in Lugol's solution five to ten minutes. 

3. Dehydrate quickly in 1 part of tincture of iodin to 3 or 
4 parts of absolute alcohol. 

4. Clear and mount in oleum origani cretici. 

The color is said to keep remarkably well. Other oils or 
balsam cause it to fade quickly. The staining in Lugol's 
solution may be omitted, as the tincture of iodin usually 
stains the amyloid sufficiently deeply. 

Iodin and Sulphuric-acid Reaction. — 1. Stain quickly 
and lightly in dilute Lugol's solution. 

2. Treat with sulphuric acid, either concentrated or dilute 
(1 to 5 per cent.), on the slide or in the staining dish. Strong 
hydrochloric acid may be used in the same way. 

The change of colors from red to blue already spoken of 
usually occurs within a few minutes, but occasionally does 
not take place at all. 

The following substances give reactions with the above 
iodin tests : 

1. Cholesterin crystals are stained rather dark with dilute 
iodin solution, and turn a beautiful blue color at the edges 
on the addition of strong sulphuric acid. 

2. The corpora amylacea in the prostate and central ner- 
vous system stain brown with the dilute iodin solution. 

3. Starch-granules stain blue with dilute iodin solution. 

4. Cellulose stains yellow with iodin. If washed and 
treated with strong sulphuric acid, it turns blue where the 
acid touches it. 

For the reactions with the aniline dyes the sections must 
be free from celloidin. 

Reaction with Methyl-violet. — 1. Stain frozen sections of 
fresh or of formaldehyde or alcohol fixed tissue in 1 per 
cent, methyl-violet three to five minutes. 

2. Wash in a 1 per cent, aqueous solution of acetic acid. 

3. Wash thoroughly in water to remove all trace of acid. 



1 82 PATHOLOGICAL TECHNIQUE. 

4. Examine in water or in glycerin. 

The stain will keep for some time if mounted in a satu- 
rated solution of acetate of potash or in levulose. Other 
methods are to stain in aniline-methyl-violet and to wash 
out in a 1 per cent, solution of hydrochloric acid, or to stain 
in a strong solution of methyl-violet to which acetic acid is 
added, and to wash out in water. The amyloid is stained 
violet-red, the tissue blue. Sections of tissues imbedded in 
celloidin will not give the reaction unless the celloidin is 
removed. The color reaction shows best when the light for 
the microscope is taken from a white cloud, not from the blue 
sky. 

Reaction with Iodin-green. — 1. Stain fresh or hardened 
sections in a \ per cent, aqueous solution of iodin-green for 
twenty-four hours. 

2. Wash in water. 

3. Mount in water or glycerin. 

Amyloid, a violet-red ; tissue, green. Stilling claims that 
the reaction is surer than with methyl-violet. 

Reaction with Bismarck Brown and Methyl-violet 
(Birch-Hirschfeld). — 1. Stain in a 2 per cent, alcoholic solu- 
tion of Bismarck brown for five minutes. 

2. Wash in absolute alcohol. 

3. Wash in distilled water ten minutes. 

4. Stain in a 2 per cent, aqueous solution of methyl-violet 
five to ten minutes. 

5. Wash in dilute acetic-acid solution. 

6. Wash thoroughly in tap water. 

7. Mount in levulose. 
Amyloid, red; tissue, brown. 
Mayer's Stain for Amyloid. 

1. Transfer paraffin sections without previous treatment 
directly from the knife to a warmed (40 C.) half per cent, 
aqueous solution of gentian-violet for five to ten minutes. 

2. Wash in water and differentiate in a I per cent, solu- 
tion of acetic acid for ten to fifteen minutes. 

3. Wash thoroughly in water. 

4. Transfer to a half concentrated aqueous solution of 
alum. Wash off in water. 



HISTOLOGICAL METHODS. 1 83 

5. Transfer sections to slide and allow the water to evapo- 
rate. 

6. Remove paraffin and clear with xylol. Mount in xylol 
balsam. 

Pigmentation. — The various pigments found in the 
human body under normal and pathological conditions may 
be divided into three groups : 

1. Hematogenous pigments, derived from the coloring 
matter of the blood. 

(a) Hemoglobin and methemoglobin : soluble in water and 
alcohol, not absolute ; occur as yellowish to yellowish-brown 
granules and droplets ; stain deeply with eosin after proper 
fixation ; occur in hemoglobinuria, etc. 

(b) Par hemoglobin : a form of hemoglobin ; crystallizes 
like it, but is insoluble in alcohol. 

(e) Hemaloidin=bilimibin : contains no iron ; is insoluble 
in water, alcohol, and ether; dissolves in chloroform ; occurs 
as yellow or brown amorphous material or as crystalline 
rhombic plates and needles. Is found in extravasations of 
blood. 

(d) Hemosiderin : occurs as bright-colored, yellowish- 
brown and brown granules and masses ; gives iron reac- 
tion; is insoluble in water, alcohol, and ether; is found in ex- 
travasations of blood, in the liver in pernicious anemia, etc. 

(e) Melanin: occurs as dark-brown or black granules and 
masses ; does not give iron reaction ; is found in malaria in 
the red blood-corpuscles and in the tissues of the spleen, 
liver, and brain, but not of the lungs. 

(/) Bile-pigment=bilirubi?i=hematoidin : insoluble in water, 
ether, and alcohol ; occurs as yellowish granules and masses 
which are often greenish if old ; is found in jaundice. 

2. Autochthonous pigments, formed by cells from color- 
less elements of nutrition. They all occur microscopically as 
lighter or darker brown granules ; are insoluble in water, 
alcohol, dilute caustic potash, etc., and contain no iron. 
Many of them are combined with fat, and hence seem to give 
some of the reactions for fat. They are found in the iris, 
retina, skin, ganglion-cells, Addison's disease, melanotic sar- 
comata, etc. 



1 84 PATHOLOGICAL TECHNIQUE. 

3. Extraneous pigments, entering the body from with- 
out. The most common examples are carbon in anthra- 
cosis pulmonum, iron in siderosis pulmonum, silver in 
argyria. 

Pigments are recognized microscopically, partly by their 
color and form, partly by their chemical reactions, and partly, 
though less accurately, by the lesions or pathologic proc- 
esses in connection with which they occur. They usually 
show best in contrast to red nuclear stains, such as alum or 
lithium carmine, but alum-hematoxylin often gives excellent 
results. 

The pigments of the second and third groups are perfectly 
preserved by all the ordinary fixatives, of which alcohol, 
corrosive sublimate, and Zenker's fluid can be particularly 
recommended. Of the first group, melanin and hematoidin 
are preserved in any fixative. Hemoglobin and methemo- 
globin must be fixed in the solutions recommended for red 
blood-globules — namely, Zenker's fluid, corrosive sublimate, 
and Miiller's fluid. Parhemoglobin and hemosiderin should 
be preserved in alcohol. Bile-pigment is turned green, accord- 
ing to Ziegler, by fixation in corrosive sublimate, and is 
thereby rendered more prominent. In alcohol it preserves 
its yellow color. Carbon may be distinguished from melanin 
or any of the other pigments by the fact that it is insoluble 
in concentrated sulphuric acid. 

Iron-containing Pigments : Hemosiderin. — The iron- 
containing pigments which give microchemical color reac- 
tions are included under the term "hemosiderin." They are 
derived from the hemoglobin of the red blood-corpuscles as 
a result of their destruction in the circulation or escape from 
the blood-vessels. The iron contained in hemoglobin does 
not react to the ordinary tests because it is too intimately 
bound up in the molecule, but when the hemoglobin is trans- 
formed into hemosiderin through the action of living cells then 
several of the iron tests are applicable both to the sections and 
to gross specimens, either in the fresh condition or after fixa- 
tion in certain fluids. The hemosiderin occurs in the form of 
yellowish-brown granules of various sizes, but other pigments 
may present the same appearance; hence the need of defi- 



HISTOLOGICAL METHODS. 



I8 5 



nite chemical tests. It is important always to obtain the 
tissue for fixation in as fresh a state as possible. 

The best general fixative for tissues containing iron pig- 
ments is alcohol. Formaldehyde is not so good, especially 
if tissues are allowed to stay in it very long. The alcohol- 
formaldehyde mixture is better. Corrosive sublimate solu- 
tion without the addition of acetic acid may be used. All 
mixtures containing chrome salts are to be avoided, as they 
delay or prevent the chemical reactions. 

Certain general precautions are necessary. In making up 
fixatives use water free from iron, because if lime salts are 
present in the tissues they readily precipitate the iron and 
thus become incrusted with it, so that later they will give the 
iron reaction. Microtome knives used in cutting sections 
must be free from rust. Iron needles cannot be used in 
transferring sections through the different solutions. Employ 
a platinum needle or a glass rod. 

The iron tests most available and useful are three in 
number : 

1. With ferrocyanide of potassium to form ferric ferro- 
cyanide of potassium (Prussian or Berlin blue). 

2. With ferricyanide of potassium to form ferrous ferri- 
cyanide of potassium (Turnbull's blue). 

3. With ammonium sulphide to form the greenish-black 
sulphide. 

The test ordinarily used is the first because the iron salts 
in hemosiderin are commonly ferric in nature, the method 
is simple, and the color effect is attractive. 

The second reaction, with ferricyanide of potassium, is 
occasionally useful, especially after a preliminary treatment 
with ammonium sulphide. 

The third reaction, with ammonium sulphide, is a mal- 
odorous procedure, the color effect is not attractive, and the 
method has the disadvantage of causing celloidin sections 
to wrinkle badly. Moreover, it gives a similar black reac- 
tion with certain other metals (silver, lead, mercury). 
On the other hand, it has the advantage of reacting with 
both the ferric and the ferrous salts, and thus of turning 
both of them black. On this account and because the pro- 
cedure is claimed to give the best and most accurate results 
in demonstrating iron, it has been found advisable to follow 



1 86 PATHOLOGICAL TECHNIQUE. 

the ammonium sulphide reaction with either the ferri- or 
the ferrocyanide reaction and thus turn the black color to 
the more attractive and distinctive blue color. Person- 
ally we have not had good results with this second step: 
the granules lose their sharpness and tend to fuse together. 
Method i. Prussian (Berlin) blue: 

1 . Fix tissues in alcohol or formaldehyde. 

2. Cut sections by the freezing or by the celloidin method. 

3. Place sections in the following freshly combined mixture : 
2 per cent, aqueous solution of ferrocyanide 

of potassium, 1 part; 

1 per cent, aqueous solution of hydrochloric 

acid, 3 parts, 

for twenty to thirty minutes or longer. If prolonged ac- 
tion is desirable, change the sections to a freshly prepared 
mixture every twenty or thirty minutes because the action 
of the solution gradually weakens. 

4. Wash thoroughly in distilled water. Prussian blue is 
somewhat soluble in tap-water. 

5. Counterstain with alum-carmine or lightly with alum-r 
hematoxylin and eosin. 

It is also possible to counterstain with lithium carmine, 
but it must be done before performing the iron reaction, 
not afterward, because the Prussian blue is slightly soluble 
in alkali. 

Method 2. TurnbulV s blue: 

1. Place frozen or celloidin sections in the following 
freshly combined mixture: 

2 per cent, aqueous solution of ferricyanide 

of potassium, 1 part; 

I per cent, aqueous solution of hydrochloric 

acid, 3 parts. 

2. Transfer to 1 per cent, hydrochloric acid for from five 
to ten minutes. 

3. Wash thoroughly in water. 

4. Mount in glycerin or pass through alcohol and oil 
and balsam. Counterstain as in Method 1. 

Method 3. Greenish black sulphide: 

1. Place frozen or celloidin sections in a concentrated, 
somewhat yellowish solution of sulphide of ammonium 



HISTOLOGICAL METHODS. 1 87 

(which should be at least twenty-four hours but not over 
three weeks old) for one-half to twenty-four hours. 

2. Wash in distilled water. , 

3. Mount in glycerin or transfer through alcohol and oil 
to balsam. The sections can be counterstained before 
mounting either with alum-carmine or lightly with alum- 
hematoxylin and eosin. 

The black color can be changed to blue by following this 
method with either Method 2 (Tirmann and Schmelzer) or 
Method 1 (Nishimura), but according to our experience the 
stains obtained in this way are not so clean cut as those 
which Method I used alone yields, provided the acid is in 
excess of the ferrocyanide of potassium. 

Petrifaction. — Calcification, the more common form of 
petrifaction, is the term applied to the infiltration of tissues 
with phosphate and carbonate of calcium. The salts appear 
microscopically as small, very refractive granules which may 
be mistaken for fat, or as large masses due to the fusion of 
granules. They are dissolved by hydrochloric or nitric acid 
(5 per cent, solution). If carbonate of lime is present, bubbles 
of carbon dioxid are set free. Phosphate of lime dissolves 
without effervescence. To differentiate between lime-salts 
and other substances soluble in hydrochloric acid use con- 
centrated sulphuric acid to form sulphate of lime (gypsum), 
which appears as fine, short, radiating needles. On dissolv- 
ing out the lime-salts a matrix of dead tissue or of hyaline 
material will usually be found left behind. As a rule, this 
hyaline material stains deep blue in alum-hematoxylin or red 
in Van Gieson's mixture. 

The deposits of calcium salts themselves also stain with 
hematoxylin, so that it can be used to demonstrate the 
masses and coarser granules of them. The tissue must, how- 
ever, first be freed of certain iron combinations, which are often 
associated with deposits of lime and also stain with hema- 
toxylin. The following method is recommended by Roehl : 

Roehl's Hematoxylin Method. — 1 . Fix in alcohol or for- 
maldehyde. 

2. Place sections in a half-concentrated solution of oxalic 
acid for fifteen to thirty minutes to remove the iron. 

3. Wash thoroughly in water. 



1 88 PATHOLOGICAL TECHNIQUE. 

4. Stain in a 1 per cent, aqueous solution of hematoxylin 
(which must be neither too fresh nor too old) for five to ten 
minutes. 

5. Differentiate in water, to which a few drops of ammonia 
water are added, until the section is colorless and only the 
lime deposits remain stained. 

6. Wash in water. 

7. Counterstain with safranin. Alcohol ; xylol ; balsam. 
Lime-salts deep violet ; nuclei red. 

Von Kossa has shown that phosphate of calcium can be 
demonstrated by means of nitrate of silver, which forms silver 
phosphate on the surface of the granules and blackens in the 
presence of light. It gives an exaggerated picture of the 
amount of lime-salts present. Klotz has shown that the 
nitrate of silver acting for many hours affects calcium car- 
bonate also : the granules become coated with silver car- 
bonate, which in sunlight gives off carbon dioxid, leaving the 
black silver oxid. This process can be hastened by putting 
the sections, after staining and thorough washing, into a 
dilute soluble sulphide. 

Von Kossa's Silver Method. — 1. Fix in alcohol, formal- 
dehyde, or corrosive sublimate. 

2. Place sections (frozen, celloidin, paraffin) in a I to 5 per 
cent, aqueous solution of nitrate of silver for thirty to sixty 
minutes (von Kossa), three to twelve hours (Klotz). 

3. Wash thoroughly in distilled water. 

4. Mount in glycerin or, after dehydration and clearing, in 
xylol balsam. 

The lime is stained deep black. The nuclei can be counter- 
stained with alum carmine or safranin after the silver staining. 

Another form of petrifaction is that found in gout, due to 
the infiltration of certain tissues with uric-acid salts, of which 
urate of sodium is the most common. The crystals are sol- 
uble with difficulty in cold water, insoluble in alcohol and 
ether. Therefore, to study the deposits in connection with 
the lesions, fix in 95 per cent, alcohol and imbed in celloidin ; 
stain sections quickly in a cold solution of alum-hematoxylin. 
Wash quickly in cold water and transfer to alcohol. Clear 
and mount in balsam. 



CULTURE-MEDIA. 



Culture-media consist of various nutritive substances, 
either liquid or solid, in or upon which bacteria will grow 
and multiply, and are, as a rule, contained in test-tubes ready 
for use. 

The nutritive material in these test-tubes must be free from 
living bacteria — i. e. " sterile " — and must be kept so until 
used. This is accomplished by inserting a stopper of raw 
cotton into the mouth of each test-tube to exclude the en- 
trance of bacteria from without, and then subjecting the 
tubes and their contents to the sterilizing action of live 
steam for the purpose of killing any bacteria which may 
have gained access to the medium during its preparation. 

The Preparation of Test-tubes. 1 — New test-tubes 
should be washed in a very dilute solution of nitric acid 
(2-5 c.c. of the commercial nitric acid to the liter of water), 
then thoroughly rinsed in water and allowed to drain until 
dry or nearly so. The object of the use of the nitric acid is 
to remove any free alkali which may be present in the new 
tubes. 

Old test-tubes containing culture-media, after removal 
of the cotton stoppers, should be boiled for from half an 
hour to one hour in a solution of common soda (4-6 per 
cent.). This treatment not only destroys bacteria, but it also 
loosens and liquefies the material in the tubes, so that it may 
be easily removed with the aid of a test-tube brush and 
plenty of water. 

When all the material has been removed from the test- 
tubes in this way, they are to be rinsed in clean water, then 
in the dilute nitric acid of the strength above indicated for 

1 Test-tubes of the size known as 6 X I in. are recommended. 

189 



I90 PATHOLOGICAL TECHNIQUE. 

the new test-tubes, and finally again rinsed in clean water, 
after which they are to be allowed to drain until dry or 
nearly so. 

The test-tubes thus prepared are next to be provided with 
stoppers of raw cotton (not absorbent cotton), which are to be 
inserted into the mouths of the tubes for a distance of about 
3 cm., and should fit the walls of the tubes smoothly. The 
stoppers should not be packed in nor fit too tightly, but be 
just firm enough in position to easily sustain the weight of 
the tube when it is lifted by the projecting portion of the 
cotton. 

The stoppered tubes are then to be packed into a square 
wire basket which fits into the hot-air sterilizer, 1 and heated 
in this, with the door closed, until the temperature reaches 
about 150 C. The object of this heating is not to sterilize 
the tubes and cotton stoppers, but to mould the stoppers to 
the shape of the test-tubes, so that they can readily be re- 
placed when removed in the subsequent filling of the tube 
with nutritive material. In packing the tubes into the square 
wire basket as many as possible should be placed with the 
cotton stopper uppermost, and the remainder of the space in 
the basket above the tubes may be filled with tubes placed 
on their sides. 



PREPARATION OF CULTURE=MEDIA. 

Bouillon. — Formula for 1000 c.c. : 

Lean beef, 500 grams ; 

Or extract of beef, 3 " 

Pepton, 10 " 

Sodium chlorid, 5 

Water, 1000 c.c. 

500 grams, or about 1 \ pounds, of lean beef, finely minced, 
are thoroughly mixed with 1 000 c.c. of ordinary tap-water 
and the mixture is then boiled in a saucepan over the gas 

1 See any dealer's catalogue of bacteriological apparatus. 



CUL TURE-MEDIA. 1 9 1 

stove 1 for about half an hour. It is next filtered through 
filter-paper to obtain the clear infusion of the beef, free from 
the coagulated albumin and shreds of tissue. This clear 
beef-infusion is then turned back into the saucepan, which 
should be clean, and to it are added 10 grams of pepton 
(Witte), 5 grams of sodium chlorid, and sufficient water to 
make the total volume of the mixture 1000 c.c. The volume 
of 1000 c.c. may be indicated with sufficient accuracy by a 
mark previously made on the inner surface of the side of the 
saucepan. The mixture is next to be boiled until all these 
substances are dissolved, stirring frequently with a glass rod, 
and is then to be neutralized, for it has a decidedly acid 
reaction from the acid of the meat. 

The neutralization is important and requires care (see also 
p. 202). The reaction required is that of a very faint alka- 
linity, as is shown by the production of a blue color on red 
litmus paper, while no change is produced on the blue 
litmus paper. In neutralizing, a 10 per cent, solution of 
caustic soda is added, a few c.c. at a time at first, and later,, 
two or three drops at a time, while the mixture is kept boil- 
ing, the reaction being tested between each addition of alkali 
after thorough stirring with a glass rod. 

The test of the reaction is best made by placing a drop of 
the mixture on a piece of litmus-paper by means of the 
glass rod and then moistening the paper at the water-faucet. 
In this way the best judgment can be formed of changes in 
the color of the paper. If the mixture becomes too alkaline,, 
dilute hydrochloric acid is to be added to correct this. 

When the proper reaction has been obtained the mixture 
is to be filtered through filter-paper into a flask, and suf- 
ficient water added to bring the volume of the filtrate up to 
1000 c.c, thus replacing the loss by evaporation. The fil- 
trate in the flask is now bouillon. If the bouillon be heated 
to the boiling-point, it will usually become more or less 

1 In the preparation of culture-media some form of gas stove is preferable to 
a Bunsen burner. 



jg 2 PATHOLOGICAL TECHNIQUE. 

clouded by a precipitate of phosphates. As a rule, subse- 
quent heatings do not cause any further precipitations. 
Therefore it is advisable, if it is desired to obtain perfectly 
clear bouillon, to steam the flask containing the freshly pre- 
pared bouillon in the steam sterilizer for about half an hour, 
and then, if the bouillon be clouded, to again filter, so that 
the subsequent sterilizations in the test-tubes will not cause 
precipitates. 

The finely minced beef may be obtained in the shops 
under the name of Hamburg steak, or it may be very readily 
prepared with the aid of a meat-grinder. 

The usual directions for the preparation of bouillon require 
that the mixture of the minced meat and water be allowed 
to stand over night in a cool place before boiling. In our 
experience this is not necessary. 

For bouillon cultures the bouillon is run into test-tubes, 
each tube being filled to a depth of about 4 cm., and steril- 
ized immediately and on the two following days, according 
to the general directions given on page 206, after which it is 
ready for use. 

Bouillon may also be made as above indicated by using 
three grams of Liebig's extract of beef to the liter, instead 
of the beef-infusion. 

Glucose or Dextrose Bouillon. — Formula : 



Glucose or dextrose (dry), 


10 grams ; 


Lean beef, 


500 " 


Or extract of beef, 


3 " 


Pepton, 


10 " 


Sodium chlorid, 


5 " 


Water, 


IOOO c.c. 



This medium is identical with the preceding, except that it 
contains 10 grams of glucose to the liter (1 per cent.) in ad- 
dition to the other ingredients. The preparation of glucose 
bouillon is the same as that of plain bouillon, the glucose 
being added with the pepton and sodium chlorid. 



CUL TURE-MEDIA. 1 93 

Agar-agar (plain). — Formula for 1000 c.c. : 



Agar-agar, 


15 grams; 


Lean beef, 


500 


Or extract of beef, 


3 " 


Pepton, 


10 " 


Sodium chlorid, 


5 " 


Water, 


IOOO c.c. 



Agar-agar is essentially bouillon in which agar-agar 'has 
been dissolved so that a transparent jelly is formed. The 
function of the agar-agar is merely to give the medium the 
property of becoming liquid when heated and solid when 
cool ; it is not nutritive. The nutritive substances are in the 
bouillon. 

To make one liter, 15 grams of agar-agar are placed in 
the clear beef-infusion, made as described on p. 190 and 
boiled for one hour in a saucepan. 1 The agar-agar dissolves 
slowly, and continuous boiling is necessary to ensure its 
subsequent filtration. Before boiling, about 200 c.c. of water 
should be added to compensate for evaporation, and later, as 
the level of the liquid falls, more water should be added 
from time to time. It is well to have some mark on the side 
of the saucepan which will indicate the level of a liter. 
When the boiling is nearly finished, 10 grams of pepton, 5 
grams of sodium chlorid, and sufficient water to make a 
volume of one liter are added to the mixture. The mixture 
is then neutralized, as described for bouillon, while still boiling. 

After the boiling is completed the saucepan is to be placed 
in cold water until the temperature of its contents falls 
to about 6o° C, as shown by the thermometer, the cooling 

1 If an autoclave (see p. 207) be available, it may be used very conveniently 
in hastening the solution of agar-agar in the meat-infusion. For this purpose 
the mixture of finely fragmented agar-agar and the beef-infusion should be 
placed in a Florence flask. When the temperature of the interior of the auto- 
clave has reached about 120 C. or when the gauge shows a pressure of 
two atmospheres, the heat should be turned off and the apparatus allowed to 
cool to about ioo° C. before opening. The mixture is then transferred to a 
saucepan and the preparation proceeded with as above indicated. 
13 



194 



PA THOL O GICA L TE CHNIQ UE. 



being facilitated by stirring with a glass rod. When this 
temperature is reached, an egg is beaten into the mass and 
the saucepan with its contents replaced on the stove, where 
it is slowly brought to boiling and boiled for about ten min- 
utes. The object of the adding of the egg is to clarify the 
medium. It is then filtered, boiling hot, through wet folded 
filter-paper into a flask. A funnel with corrugations on its 
sides is best to use. With this the folding of the filter-paper 
is not necessary. 

In order to save time, it is best to use two filters and two 
flasks at once, for the filtration rapidly becomes slow as the 
mass cools, and several heatings of the residue on the filter 

are necessary. As soon as the fil- 
trate begins to appear slowly, drop 
by drop, the mass remaining on 
the filter should be turned back 
into the saucepan — which can best 
be done by making a hole in the 
bottom of the filter with the glass 
rod — and brought again to boiling. 
While boiling hot it is again poured 
on a fresh filter. This preparation 
of fresh filters and reheating may 
have to be repeated several times 
before all of the mixture is filtered. 
The filtration may also be carried 
on in the steam sterilizer to pre- 
vent the cooling of the medium. 

When the amount of coagulated 
egg-albumin and medium remain- 
ing on the filter does not exceed a 
volume of 50-100 c.c, the filtra- 
tion may be considered complete. 
To the filtrate, which is now agar- 
agar, is next added sufficient water 
to make up the loss by evaporation, 
and the medium is then to be run 
into test-tubes and sterilized, as described on page 206. 




FlG. 14.—" Stab " culture 
" slant" culture (£). 



(«) 



CUL TURE-MEDIA. 1 9 5 

In view of the difficulty of filtering agar-agar, it has been 
proposed to avoid this operation by placing the fluid medium 
in a sedimenting vessel, such as a large funnel, with closed 
apex. The solid particles settle to the bottom if the medium 
be maintained in a fluid condition in the steam sterilizer for 
a certain length of time. When the medium has become 
solid it is turned out of the vessel as a cast, and the bottom 
portions, containing the sediment, cut off from it and rejected. 
The remaining portion will be found clear enough for most 
purposes and may be melted up at once for distribution in 
tubes, or if it now be desirable to further clarify it, it may be 
melted up and filtered as described above. It will be found 
to filter more readily than before. 

Precipitates of phosphates in the medium frequently occur 
after the first sterilization, but if these be removed they do 
not usually appear again in subsequent heatings. Therefore, 
if it be desirable to obtain a very clear agar-agar, it is well to 
place the flask containing the freshly-prepared medium in 
the steam sterilizer for half an hour, and then filter again to 
remove any precipitate which may have appeared. The 
subsequent sterilization in the test-tubes will then cause no 
precipitation. 

In filling the test-tubes it is customary to fill some tubes 
to a depth of about 3 cm. and others to a depth of about 5 
cm. After the complete sterilization of the medium in the 
tubes as described on p. 206, the first-mentioned tubes are 
placed on their sides with their mouths slightly elevated 
while the medium is still fluid, so as to form, after solidification, 
a slanting surface extending from near the bottom of one 
side of the tube to about half the length of the tube on the 
opposite side. The solidification of the agar-agar takes place 
in a short time, and as soon as it occurs the tubes are ready 
for use, this form of culture being known as a " slant " tube 
or culture (Fig. 14, b). It is well, however, to allow the 
tubes to remain in their slanting position for a day or two to 
permit the medium to become more or less adherent to the 
walls of the tube, and thus avoid its tendency to slide down- 
ward when the tubes are placed in the upright position. 



196 PATHOLOGICAL TECHNIQUE. 

The tubes filled to a depth of 5 cm. are to be allowed to 
cool and solidfy while in an upright position, and the form 
of culture-tube thus obtained is called a " stab " culture (Fig. 
14, a), because the medium in the tube is inoculated for cul- 
ture purposes by inserting an infected platinum wire into its 
depths. 

Glucose Agar-agar. — Formula for 1000 c.c. : 



Glucose (dry), 


10 grams; 


Agar-agar, 


15 " 


Lean beef, 


500 " 


Or extract of beef, 


3 " 


Pepton, 


10 " 


Sodium chlorid, 


5 " 


Water, 


IOOO c.c. 



This medium differs from plain agar-agar only in the addi- 
tion of 10 grams (1 per cent.) of glucose. The glucose 
should be obtained in the form of solid masses, not as a thick 
fluid, and it is to be added with the pepton and sodium 
chlorid. In short, glucose agar-agar is made with glucose 
bouillon in identically the same manner that plain agar-agar 
is made with plain bouillon. 

Lactose- litmus agar-agar consists of plain agar-agar to which 
has been added 2 or 3 per cent, of lactose and sufficient litmus 
tincture to give it a pale-blue color. 

Glycerin Agar-agar. — Formula for 1000 c.c. : 



Glycerin, c. p., 


60 c.c. ; 


Agar-agar, 


15 " 


Lean beef, 


500 grams ; 


Or extract of beef, 


3 " 


Pepton, 


10 " 


Sodium chlorid, 


5 " 


Water, 


IOOO C.C. 



This medium is prepared by adding to plain agar-agar after 
its final filtration, and before running it into the test-tubes, 60 
c.c. (6 per cent.) of glycerin c. p., and mixing thoroughly. 



CUL TURE- MEDIA. 1 97 

Blood Agar. — Sterile defibrinated blood is mixed with 
fluid agar (made from meat infusion) in the proportion of 
I to 2 or 3, and "slants" or "Petri plates" prepared with 
the mixture. 

The blood of man and various animals may be used. 
It may conveniently be drawn from a vein with a syringe; 
in the case of the rabbit, from the heart. 

Of course, sterile precautions must be observed. 

Gelatin (plain). — Formula for iooo c.c. : 



Gelatin, 


IOO grams 


Lean beef, 


500 " 


Or extract of beef, 


3 " 


Pepton, 


10 " 


Sodium chlorid, 


5 " 


Water, 


1000 c.c. 



Gelatin is essentially bouillon in which gelatin has been 
dissolved, so that a transparent jelly is produced which is 
solid at ordinary temperatures and fluid when slightly 
warmed. To prepare one liter, 100 grams (10 per cent.) 
of golden seal French gelatin are dissolved in a liter of the 
hot bouillon which has been heated to boiling in a saucepan. 
When the gelatin is thoroughly dissolved the mixture is 
boiled for about five minutes, and the marked acidity of the 
gelatin then carefully neutralized by the addition of caustic 
soda, in 10 per cent, solution, to a very faint alkalinity, as 
has been described in the preparation of bouillon. As in 
the case of agar-agar, the mass is then cooled to 6o° C, an 
egg beaten into it, then gently heated again to boiling, and 
boiled about ten minutes, when it is to be filtered through a 
wet folded filter into a flask. Gelatin usually filters fairly 
rapidly, but time may be saved by using two filters at once. 
When filtered it is to be run into test-tubes and sterilized, 
as described on page 206. It is used both in the form of 
" slant " and " stab " cultures, as in the case of agar-agar 
(see page 195). 

In the preparation of this medium it is important to sub- 
ject it as little as possible to the boiling temperature, for 
prolonged exposure to this destroys its power of solidifying. 



r 9 8 PATHOLOGICAL TECHNIQUE. 

Therefore in sterilizing, gelatin tubes should never be allowed 
to remain exposed to live steam longer than twenty minutes. 
It is also important to apply the heat slowly during the pro- 
cess of heating after the addition of the egg above mentioned, 
in order to avoid " burning." 

Glucose or Dextrose Gelatin. — Formula for iooo c.c. : 



Glucose or dextrose, 
Gelatin, 


10 grams ; 
IOO " 


Lean beef, 

Or extract of beef, 
Pepton, 
Sodium chlorid, 


500 

3 " 
10 

5 " 


Water, 


1000 c.cm. 



This medium is essentially gelatin dissolved in glucose 
bouillon (see page 192), and is prepared in the same manner 
as the plain gelatin, except that glucose bouillon is used in- 
stead of plain bouillon. 

Blood-serum (Loffler's Mixture). — Formula : 

Glucose bouillon (see p. 192), 1 part; 
Beef blood-serum, 3 parts. 

This culture-medium consists of a mixture of the blood- 
serum of the bullock and glucose bouillon, which is run 
into test-tubes and coagulated by heat in such a way as to 
form a slanting surface for culture purposes — i. e. it is used 
in the form of "slants." 

The blood-serum is collected at the slaughter-house in tall 
glass jars of the capacity of a gallon or more. These jars 
should be thoroughly clean, but sterilization is not necessary. 

The blood which is obtained by the Jewish method of 
slaughter — viz. by severing the carotid artery — is the best for 
the purpose, because it clots more readily. As the blood 
runs from the vessels of the animal it is received in the glass 
jar, and immediately placed in a cool place for twenty-four 
to forty-eight hours to allow it to clot and the serum to 
separate. All unnecessary agitation of the fresh blood 
should be avoided, as this interferes with its proper clotting. 
It is well to inspect the blood after a few hours, and gently 



CUL TURE-MEDIA . 1 99 

loosen with a clean glass rod any adhesions which the clot 
may have formed to the wall of the jar, thus allowing the 
clot to more readily contract and squeeze out the serum 
from its meshes. After about twenty-four hours the serum 
is removed by the aid of a clean pipette and brought to the 
laboratory. If the clot is in good condition, more serum will 
appear after another twenty-four hours, and if necessary this 
also may be used. 

The presence of red blood-corpuscles in the serum is of 
little importance. Three parts of the beef blood-serum thus 
obtained are to be thoroughly mixed with one part of glucose 
bouillon (vide supra), convenient quantities being 900 c.c. of 
blood-serum and 300 c.c. of glucose bouillon. 

This mixture is then run into test-tubes as described on 
page 204. The quantity run into each test-tube should be 
sufficient to fill it to a depth of about 3-4 cm. The tubes 
containing the requisite amount of the mixture are next 
subjected to the action of heat while in a slanting posi- 
tion, so that the mixture in the tubes may become solid 
or coagulated, and so offer a smooth slanting surface for 
culture purposes extending from a point near the bot- 
tom of the tube to about halfway up the opposite side or 
higher. 

The coagulation is effected either in the hot-air sterilizer 
by packing the tubes on their sides, the proper slant being 
secured by means of strips of cardboard placed between the 
layers of tubes, or better, in the blood-serum coagulator 
which may be obtained from dealers in bacteriological 
apparatus. 

If the hot-air sterilizer is employed, the temperature should 
not exceed 90 C. nor fall below 85 ° C, and the door should 
be kept closed. It is optional whether the sterilizer be 
packed full of tubes or only a few layers of tubes be coagu- 
lated at a time, with careful watching to avoid overheating. 
In the former case two or three hours will be required to 
firmly coagulate the tubes in the middle layers, while the 
lower layers may be overheated. To avoid this overheating 
of the lower layers, a false bottom or one or two layers of 
empty tubes may be employed. 



200 PATHOLOGICAL TECHNIQUE. 

The blood-serum coagulator is much more convenient and 
gives much more satisfactory results. The temperature 
of the interior should be kept at about 95 ° C. To save 
time in heating, the apparatus may be filled with hot water 
from the hot-water faucet. 

Whichever apparatus is employed for coagulation, it is of 
the utmost importance that the coagulation of the mixture 
be a thorough one, and that the medium in the tubes becomes 
firm and solid, otherwise bubbles and cavities will form in it 
and destroy its smooth surface when it is subjected to the 
subsequent steam sterilization. When the tubes are firmly 
coagulated they are to be packed with the cotton stopper 
uppermost in a round wire basket and sterilized by steam 
three times, as indicated on page 206, after which they are 
ready for use. 

This method of preparing blood-serum tubes is very 
different from the one usually described, a most tedious 
and time-consuming procedure, requiring a high degree of 
technical skill, by which it is practically impossible to make 
use of blood-serum tubes for ordinary purposes. 

With the method here detailed we think that the best cul- 
ture-medium for the routine examination of pathological 
material is obtained. It is preferred by us for various 
reasons, chief of which are as follows : 

First, the ease and facility with which it can be prepared, 
especially when a proper coagulating apparatus is available. 

Secondly, the greater and more rapid growth of certain 
important pathogenic bacteria upon it than upon ordinary 
media. 

In the method usually described the serum (which should be 
clear or free from blood-corpuscles) is obtained under all aseptic 
precautions, is carefully mixed with sterile glucose bouillon in the 
proportions given above, and the mixture then run into sterile 
test-tubes. During all the manipulations precautions are neces- 
sary to avoid contamination, the serum never being allowed to 
come in contact with any object which is not sterile, and exposure 
to the air during the processes of transference from one vessel to 
another avoided as much as possible. 

The mixture now being in test-tubes, it is subjected for one 
hour on each of five successive days to a temperature of 68° to 
70 C. in a chamber provided with a water-jacket. This tern- 



CUL TURE- MEDIA. 20 1 

perature is sufficient to kill the vegetative forms of any bacteria 
which may be in it, but does not coagulate the medium. The 
intervals between the sterilizations are for the purpose of allowing 
any spores to develop into the vegetative form and thus become 
susceptible to the destructive action of heat. 

After the fifth sterilization the medium is solidified in the tubes 
in the form of " slants " by slowly raising the temperature of the 
chamber to about 8o° C, and keeping the tubes at this tempera- 
ture for several hours. In solidifying the great object is to obtain 
a gelatin-like, fairly transparent medium and to prevent opacity. 
To attain this it is necessary to proceed very carefully with the 
heating and avoid overheating or too rapid heating, the tubes 
being inspected from time to time and removed from the chamber 
as soon as their contents have the proper consistency. When 
gelatinized the tubes are placed in the incubator for twenty-four 
hours to determine whether they are sterile, after which they are 
ready for use. 

The blood-serum medium produced by this older method is 
especially suited for the cultivation of certain pathogenic bacteria 
— for instance, the bacillus tuberculosis and the bacillus diphtheriae 
— but we do not think that its superiority in this respect over the 
more readily prepared, firmly coagulated form above described is 
sufficiently marked to compensate for the great difficulties in its 
preparation. 

Litmus-milk is a form of culture-medium used for deter- 
mining certain of the physiological properties of bacteria. It 
consists of cow's milk which has been colored blue by litmus 
and containing a minimum amount of cream. A pint or so 
of strictly fresh milk is placed in a flask and steamed in the 
steam sterilizer for about half an hour. When it is removed 
it will be found that most of the cream has collected at the 
surface, and it is then easy to draw off the milk from the 
deeper layers with a pipette into a separate flask. To the 
milk from which most of the cream has been thus removed 
is added sufficient of an aqueous solution of litmus (freshly 
filtered) to give it a pale-blue color. The colored milk is 
then run into test-tubes (5 cm. deep in each tube) and ster- 
ilized, as indicated on page 206, after which it is ready for 
use. It is of great importance that the milk be fresh. If 
it is not, it may contain spore-bearing bacilli which it is 
practically impossible to kill by the steam sterilization. 

Potato-cultures according" to Bolton. — Potatoes — pref- 



202 



PATHOLOGICAL TECHNIQUE. 





erably old ones — are first washed to remove all the coarser 
particles of soil, and then solid cylinders are cut out of them 
with a cork -borer or apple-corer. These cylinders should 
be of a suitable diameter to fit into the test- 
tubes used for other culture-media, and should 
be about 5 cm. long. They are then cut 
longitudinally in an oblique direction with a 
sharp knife, so that a smooth slanting sur- 
face is produced, beginning near one end 
and extending diagonally to the other end. 
The pieces of potato thus prepared are next 
to be washed in running water over night. 
After washing, each piece is placed in a test- 
tube, the larger end resting on the bottom 
of the tube, a few drops of water being added 
to prevent drying, and then sterilized as in- 
dicated on page 206. If desired, a small piece 
of glass rod may be placed in the bottom of 
the tube to elevate the potato above the few 
drops of water (Fig. 15). 

Dunham's Pepton Solution. — Formula 
for 1000 c.c. : 




Fig. 15. — Potato- 
culture. 



Pepton, 

Sodium chlorid, 
Distilled water, 



10 grams; 

5 " 
1000 c.c. 



The pepton and sodium chlorid are dissolved 
by boiling and the mixture filtered. The clear filtrate is 
then run into test-tubes, each test-tube being filled to a 
depth of 5 cm., and is to be sterilized as indicated on page 
206, after which it is ready for use. 

The Adjustment of the Reaction of Culture-media 
by Titration. — Because comparatively small variations in 
the reaction of culture-media may have a marked effect upon 
the morphology and mode of growth of bacteria grown 
upon them, a more exact adjustment of their reaction than 
is possible with litmus paper is desirable. This is especially 



CULTURE-MEDIA. 203 

important for media used for the cultivation of the bacteria 
of water, of soil, and of the air. For ordinary purposes of 
cultivation of bacteria, especially of the pathogenic forms, 
the adjustment of the reaction with litmus paper, as else- 
where described, if carefully done, will be found to be suf- 
ficient. 

The more exact method of adjusting a reaction is one of 
titration with phenolphthalein as an indicator. The method 
is as follows : When the culture-medium, whether it be 
bouillon, agar-agar, or gelatin, has been neutralized with the 
aid of litmus paper, made up to the proper volume, and 
when it is all ready for filtering, as described elsewhere, 5 c.c. 
of it are transferred by means of a pipette to a 6-inch por- 
celain evaporating dish ; to this 45 c.c. of distilled water are 
added, and the 50 c.c. of fluid are boiled for three minutes over 
a flame to expel any carbon dioxid which may be present. 

Next, I c.c. of a 0.5 per cent, solution of phenolphthalein in 
50 per cent, alcohol is added to the mixture in the dish, and 
immediately after this enough of a twentieth normal solution 
of sodium hydroxid is cautiously run into the dish, from a 
burette, to produce a pink color in the mixture. The judg- 
ment of the proper color which indicates that sufficient 
alkali has been run in requires some practice. The color to 
be obtained is a bright pink. The appearance of the proper 
pink color is preceded by a pinkish darkening of the fluid 
which may deceive the inexperienced. 

The quantity of the twentieth normal sodium hydroxid 
solution required to effect this result is then read off from 
the burette. The number of cubic centimeters required 
denotes the percentage by volume of a normal solution of 
sodium hydroxid which would be required to make the total 
volume of culture-medium neutral to phenolphthalein. That 
this is so will be apparent after a simple calculation. 

The reaction recommended by the Bacteriological Com- 
mittee of the American Public Health Association as a 
standard to which culture-media should be adjusted is such 
that 1.5 per cent, of a normal solution of sodium hydroxid 
would be required to be added to the medium to make it 



204 PATHOLOGICAL TECHNIQUE. 

neutral to phenolphthalein. This reaction corresponds 
closely to a faint alkalinity toward litmus, for the neutral 
point of phenolphthalein is not identical with that of litmus. 
The adjustment of the reaction to this standard is effected by 
adding to the bulk of the culture-medium sufficient normal 
sodium hydroxid solution or normal hydrochloric acid solu- 
tion. 

For example : If the titration shows that 5 c.c. of the 
medium requires 1.9 c.c. of the twentieth normal solution of 
sodium hydroxid to make it neutral to phenolphthalein, then 
the total mass of the medium will require the addition of 
1.9 per cent, or 19 c.c. for a liter, of a normal solution of 
sodium hydroxid to make it neutral ; but the reaction re- 
quired is such that 1.5 per cent, of a normal solution of 
sodium hydroxid should be required to make it neutral. 
Therefore, 0.4 per cent, or 4 c.c. for a liter, of a normal 
solution of sodium hydroxid should be added to the main 
mass of the medium. 

When the calculated amount of normal solution has been, 
thoroughly mixed with the medium and the latter boiled for 
a few minutes, the titration should be repeated as above 
described. If the desired reaction is not found to be present, 
then further adjustment by addition of the calculated amount 
of normal acid or alkali solution should be made. It is not 
to be expected that the first addition to the medium of the 
calculated amount of normal solution will give exactly the 
required reaction in every case. This is due to unknown 
side reactions which take place in the culture-media. 

When the reaction has been sufficiently adjusted, the 
medium is to be filtered and is then ready to be distributed 
in test-tubes. 

The methods of making the normal and twentieth normal 
solutions required may be found in standard works on chem- 
istry. If one has not some knowledge of chemistry, he 
would better have the solutions made by a chemist. 

The filling of the test-tubes with the fluid culture- 
media described in this section is best effected by means of 
a funnel of a capacity of about a liter. In this the fluid me- 



CUL TURE-MEDIA. 



205 



dium is placed, and by means of a pinch-cock the requisite 
quantity of medium is run into each test-tube. In running 
the medium into the test-tubes the left hand holds the test- 
tube while the right hand removes the cotton stopper and 
manipulates the pinch-cock (Fig. 16). Care should be exer- 
cised not to allow any of the medium to come in contact with 
the neck of the test-tube, for it will make the cotton stopper 




Fig. 16. — Method of filling test-tubes with culture-medium (Warren). 



stick to the walls of the tube. To avoid this, the delivery- 
tube of the apparatus should be inserted some distance into 
the test-tube in filling. 

The quantity of culture-medium run into each test-tube 
varies according to the form of culture desired and the cha- 
racter of the medium. In the case of liquid media and solid 
media designed to be used in the form of " stab " cultures the 



206 PATHOLOGICAL TECHNIQUE. 

tubes should be filled to a depth of 5 cm. For " slant " cul- 
tures of solid media a depth of about 3 cm. is sufficient, or 
enough to give a slanting surface from the bottom of the 
tube to about halfway up the opposite side. 

Small Ehrlenmeyer flasks are sometimes used for bouillon 
cultures. These are of about 100 c.c. capacity, and are filled 
to a depth of about I cm. with the medium. The necks are 
provided with cotton stoppers, and the whole sterilized and 
treated as test-tube cultures. 

STERILIZATION OF CULTURE=riEDIA. 

In general, the sterilization of culture-media is effected by 
allowing them to remain exposed to the action of live steam 
in the steam sterilizer for twenty to forty-five minutes on 
three successive days. The period of exposure to live steam 
varies somewhat with the kind of culture-medium. A single 
exposure for the time mentioned is sufficient to destroy all 
bacteria present in what is called the vegetative or non-re- 
sistant form, but it will not kill spores, which represent a 
stage in the life-history of certain bacteria, in which form the 
organism is highly resistant to sterilizing agents. 

Under favorable conditions, such as are to be found in 
culture-media at ordinary room-temperature, these spores 
develop into the vegetative or non-resistant form, which 
are easily destroyed by heat. Therefore, in order that 
the culture-medium be made sterile, it is necessary that it be 
again subjected to the action of steam on the following day 
for the same length of time, when the vegetative forms of 
the few surviving spores will have developed, and will be 
capable of destruction by ordinary exposure to live steam. 

As a further precaution a third similar sterilization on the 
next day is necessary. Therefore, three steam sterilizations, of 
from twenty minutes to one hour each, on successive days, are 
required to keep culture-media sterile for an indefinite period. 1 

1 As has been pointed out by Theobald Smith, this intermittent sterilization 
at ioo° C. may not be sufficient in some cases to kill all the spores, 
because the condition in the media may not be favorable for their development 
into vegetative forms, between sterilizations. This seems to be especially true 



CUL TURE-MEDIA. 20*J 

A freshly prepared culture-medium must be sterilized on 
the same day that it is prepared, or by the next day it may 
be found to contain living bacteria, especially if kept over 
night in a warm room. 

For the purpose of sterilization the test-tubes containing 
the media are to be placed in a round wire basket which fits 
into the steam sterilizer, 1 thus facilitating the handling of the 
tubes and also keeping them upright. 

If the medium be in a flask ready for running into test- 
tubes, and if it be not convenient to do this the same day, 
the medium may be preserved as long as desired by insert- 
ing a cotton stopper into the mouth of the flask and then 
sterilizing as above indicated. 

The time of each sterilization for bouillon, agar-agar, 
blood-serum, etc. may be fixed at half an hour ; for potato- 
culture tubes and for litmus-milk, forty-five minutes. 

In the case of gelatin, however, the time of exposure to 
live steam should be shorter, owing to the danger of destroy- 
ing the solidifying power of the medium by too much heat- 
ing. Twenty minutes' exposure is sufficient. 

Large quantities of culture-media contained in flasks 
should be sterilized for forty-five minutes to an hour, for 
obvious reasons. 

The sterilization of culture-media may also be effected in 
an autoclave. This is a steam-tight chamber for sterilizing 
by steam under pressure. Various forms of this apparatus 
are on the market. The great advantage of the use of this 
apparatus is that a single sterilization is sufficient. Exposure 
of culture-media in tubes, of glassware, and of other apparatus, 
in it to a temperature of I io° C. (6 lbs. pressure) for fifteen 
minutes suffices for sterilization in most cases. For the 

of certain anaerobic spore-producing bacilli. Such spores may be the source of 
contamination of the culture-medium when it is placed under anaerobic condi- 
tions, or when the medium is used for anaerobic cultures, because strictly ana- 
erobic bacteria may grow in company with other bacteria under aerobic con-, 
ditions. Therefore, in those cases in which it is important to be certain of the 
absolute sterility of the culture-medium, sterilization in the autoclave (q. v.) is 
necessary. 

J The "Arnold Steam Sterilizer" No. 5 is recommended. 



2o8 PATHOLOGICAL TECHNIQUE. 

sterilization of culture-media in bulk, about thirty minutes at 
this temperature is necessary. 

In using the autoclave it is requisite that the confined air 
be replaced by superheated steam. To insure this, the time 
of sterilization should be reckoned only from the time when 
the theoretical temperature, as registered by the pressure- 
gauge, corresponds with that recorded by the thermometer. 1 

The Storage of Culture-media.— In order to prevent 
evaporation and the invasion of moulds, the cotton stopper 
should be cut off close to the mouth of the tube or flask, the 
surface of the stopper well singed with a flame, and the 
mouth of the tube or flask tightly closed with a cork. 

Immediately before insertion, the portion of the cork that 
enters the tube or neck of the flask should be charred in a 
flame. If thought desirable, the cork may be sealed with 
paraffin. 

bacteriological Committee Report, Jour. Amer. Pub. Health Assoc, Jan., 
1898. 



CULTURE METHODS. 



The bacteriological examination of material obtained from 
the individual during life or at autopsies should determine 
whether bacteria are present or not, and if present their 
species and comparative number. At autopsies the exam- 
ination should also determine the extent of the distribution 
of any infecting bacteria throughout the principal internal 
organs. 

This is accomplished chiefly by means of two methods of 
examination — viz., the direct examination with the microscope 
of cover-glass preparations, and the results of cultures made 
from the tissues. Both of these methods should be em- 
ployed together, but the culture method is perhaps the most 
important. A third but less frequent method is the inocula- 
tion of animals with pieces of tissue or material taken from 
the body. 

Methods of Collecting Material. — In the bacterio- 
logical examination of pathological material obtained from 
the individual during life, it is of obvious importance that the 
material be protected from the invasion of bacteria from 
without, and that in its collection every object with which it 
comes in contact be free from living bacteria. 

To fulfil these requirements the material may be con- 
veniently collected in any of the following ways : 

1. It may be obtained directly from the individual by 
means of the sterilized platinum wire, and cover-glass prep- 
arations, cultures, and, if necessary, animal inoculations, 
made at once. 

2. Since a very small quantity of the material usually 
suffices for the purposes of examination, it may often be 
very conveniently collected and brought to the laboratory 

14 209 



210 



PA THOL GICAL TE CHNIQ UE. 



on the so-called "swabs," where it can be subjected to the 
various manipulations at leisure. 

The "swab" consists of a piece of rather stiff wire about 
six inches long, on one end of which is firmly twisted a 
pledget of absorbent cotton, so that the end of the wire is 



VsAW 





Fig. 17. — Sterilized test-tube 
and swab for collecting pus and 
fluids for bacteriological examina- 
tion (Warren). 



Fig. 18. — Apparatus for the collec- 
tion of pathological fluids. 



well covered. This is placed, cotton end first, in a test- 
tube, which is then provided with a cotton stopper (Fig. 
17), and the whole sterilized in a hot-air sterilizer by heat- 
ing to 150 to 180 C. during about half an hour. A large 
number of "swabs" in test-tubes may be kept on hand ster- 
ilized and ready for use. 



CULTURE METHODS. 211 

When it is desired to secure material for bacteriological 
examination on a "swab," the cotton stopper is removed, 
the swab taken out, and the cotton end brought in contact 
with the pus or exudate in such a manner that some ad- 
heres to the cotton. The swab is then immediately replaced 
in the test-tube, the cotton-stopper returned to its place, 
and the whole then carried to the laboratory. 

In these manipulations care should be taken to avoid 
touching with the swab anything but the material which it 
is desired to examine, otherwise the material may be con- 
taminated with other bacteria than those originally present 
in it. 

By means of swabs material for examination from pus or 
exudates may be secured and brought to the laboratory in 
most instances. They are especially useful in surgical work, 
in which it is often desirable to determine the character of 
the organism present in a pus-formation or exudation with- 
out waiting to summon a bacteriologist or to collect the 
necessary cover-glasses, culture-tubes, platinum needle, etc. 
The swabs and their test-tubes may be kept on hand in a 
sterile condition, so that they may be handled by the ope- 
rator or an assistant. 

3. Fluid material may be collected by aspiration or other- 
wise. In the case of fluids care should be taken that every- 
thing with which the fluid comes in contact be clean and 
sterilized by heat if possible. The use of antiseptics, 
such as carbolic acid or corrosive sublimate, is to be 
avoided. 

If a hypodermic syringe is used in obtaining material, it 
should be of a construction which will admit of sterilization 
by heat, and it should be so sterilized before using. 

In the collection of pathological fluids, especially peri- 
toneal exudates, a special form of apparatus has been found 
most useful. It consists essentially of a glass tube, about 
14 cm. long and about 7 mm. in external diameter, one end 
of which is narrowed to a small opening and rounded off, 
while to the other end is attached a small rubber bulb like 
that on a" medicine dropper." It is to be kept ready for 



212 PATHOLOGICAL TECHNIQUE. 

use in a test-tube, stoppered with cotton (Fig. 18), the 
whole having been sterilized as are surgical dressings. The 
rubber bulbs are not expensive. Any number of pieces of 
this apparatus may be kept on hand in sterile condition. 

When it is desired to obtain a sample of peritoneal or 
other fluid for bacteriological examination, the apparatus is 
removed from the test-tube and the fluid aspirated into it by 
manipulation of the rubber bulb. It is then replaced in the 
test-tube. The fluid thus obtained should be free from con- 
tamination and may be readily transported to the laboratory 
for examination. 

Examination by Cultures.— The demonstration of the 
presence of bacteria in a tissue or exudate by means of cul- 
tures consists in bringing a small amount of the material to 
be examined in contact with some solid nutrient substance 
in which the bacteria will thrive. On this the bacteria by 
multiplication form masses or colonies visible to the naked 
eye, and present appearances which enable a practised eye 
in many cases to recognize the species of the bacteria of 
which they are composed. Of the solid culture-media de- 
scribed in the preceding section, the coagulated blood-serum 
is distinctly the best to use for the demonstration of the 
presence of bacteria in routine pathological work, because 
certain of the most important pathogenic bacteria grow bet- 
ter upon it than upon agar-agar or similar media. The other 
media have important uses in the study of the bacteria after 
their isolation from the tissues, and in certain instances spe- 
cial media are to be used, as will be pointed out in the fol- 
lowing pages. The blood-serum medium here described 
has been found entirely suitable for the isolation of the 
bacillus tuberculosis from tubercular lesions, which proves 
its efficiency as a culture-medium. 

Method of Preparing" Cultures on Blood-serum. — The 
preparation of cultures on the coagulated blood-serum con- 
sists in distributing over the surface of the medium in a test- 
tube as much of the tissue or other material as will adhere 
to the end of a piece of stiff platinum wire hammered flat 
at the end. The wire is fixed in the end of a glass or metal 






CULTURE METHODS. 21 3 

rod, and should be about 8 cm. long. It should have a 
rounded spatula-like extremity, and should be thick enough 
not to bend easily. In making cultures from clinical mate- 
rial, the platinum loop may be used for fluids. 

The " platinum wire" or "loop" consists of a piece 
of platinum wire of about 22 gauge, 2^ to 3 inches long, 
fixed in the end of a small glass or metal rod 8 or 10 inches 
long. It is often of great convenience to have two- of these 
instruments, one with the wire curled into a simple loop 
about 1 to 2 mm. in diameter at the free end, and the other 
a straight wire with the free extremity hammered flat into 
a very small spatula. The latter is of great utility in pick- 
ing up minute portions of bacterial colonies. 

Both this instrument and the stiffer wire, above mentioned, 
should be heated to a red heat in a flame immediately before 
using, in order to destroy any bacteria that may be upon 
them. 

If the material is on a " swab," the surface of the blood- 
serum or other media may be conveniently inoculated directly 
by gently rubbing the swab over it. In this case it is usually 
best to make a dilution or two by means ol the platinum wire, 
as described below, especially if there be a large amount 
of material on the swab or if the cover-glass examination 
has shown that a large number of bacteria are present. In 
any case it is important that the infected material be spread 
over all of the surface of the medium, and not in the form 
of one or two narrow streaks. 

It -is, of course, essential that the material brought in con- 
tact with the culture-medium should be free from bacteria 
not originally present in it, or that it be not contaminated 
with bacteria from outside sources. Therefore, in taking 
material from the interior of organs and tissues the surface 
is first sterilized by searing it with a hot knife, such as an ordi- 
nary case-knife, which has been heated in the Bunsen flame, 
and then, through a small incision made with another hot 
knife in this seared or sterilized area, the material from the 
interior is collected on the end of the platinum wire, which 
has also been previously heated in the Bunsen flame to 



214 PATHOLOGICAL TECHNIQUE. 

sterilize it, and then cooled either by plunging it in the 
water of condensation of the culture-tube for a few seconds 
or by moving it about in the interior of the tissue. 

In the case of exudations on free surfaces, however, this 
searing is impossible, and therefore care should be exercised 
at the autopsy not to contaminate any such exudate by 
handling before the material for culture has been obtained 
with the platinum wire. The material thus secured is then 
transferred by means of the platinum wire to the surface of 
a blood-serum culture-tube, and the infected wire gently 
rubbed over all of the surface of the culture-medium, avoid- 
ing, however, the breaking of the surface. It is important 
that the material be well distributed over the nutrient surface. 
If the material is suspected of containing a large number of 
bacteria, as in the case of suppurations or acute inflamma- 
tory lesions, a second tube should be inoculated from the 
first one by touching the platinum wire, previously sterilized 
and cooled, to the infected surface of the first tube, and then 
gently rubbing the infected wire over the surface of the 
second tube. This oper-ation is called " diluting." The 
object of this is to obtain, after the development of the cul- 
ture, a sufficiently small number of colonies in the second 
tube, so that they may be discrete — i. e. y separated from one 
another — and thus be enabled to exhibit their characteristic 
appearances, which are largely lost when the colonies are so 
numerous as to be confluent. 

If thought desirable, a third tube may be similarly inocu- 
lated from the second, but this is rarely necessary. In mak- 
ing these " dilutions " it is well to cool the platinum wire in 
the water of condensation of the sterile tube before touch- 
ing it to the infected surface of the other tube. As a rule, 
one tube will be sufficient to obtain discrete colonies from 
organs or tissues in which no suppurative or exudative con- 
dition is present. 

Anaerobic cultures are indicated in certain cases. For 
anaerobic methods, see pp. 220, 221. 

After the manner above indicated cultures are to be made 
at the autopsy as a matter of routine from the blood of the 



CULTURE METHODS. 21 5 

heart, from the liver, the spleen, the lung, and the kidney. 
Cultures are also to be made from any acute inflammatory 
lesion in any situation. 

As each culture-tube is infected it is to be labelled with 
the name of the organ or of the material from which it was 
infected, and with the date. For this purpose small paper 
labels coated with mucilage are used. 

The culture from the blood of the heart should be made 
before the removal of that organ from the body, by searing 
the right ventricle and then puncturing it with a sterilized 
knife to admit the platinum wire. The amount of blood 
used for the culture should be as much as will adhere to the 
platinum wire. Cultures from vegetations in acute endo- 
carditis are not usually of much value unless they are suf- 
ficiently large to enable a sterilization of their surface to be 
effected and material for culture secured from their interior. 

Most pathogenic bacteria grow best at body temperature. 
Therefore, cultures in most instances are placed in an incu- 
bator and examined after twenty-four hours, or when the 
identity or diagnosis of the bacteria whose colonies have 
grown out upon them is to be established. The identifica- 
tion of the infecting bacteria present in most cases may be 
made from a consideration of the size, color, and general 
appearance of the colonies as they appear on the surface of 
the blood-serum when taken in connection with the mor- 
phology of the bacteria composing them. In some instances, 
however, this may not be sufficient evidence upon which to 
base the diagnosis, and it may be necessary to obtain further 
facts in regard to a given organism in order to identify it 
with a sufficient degree of certainty. Thus it may be neces- 
sary to observe the appearances of its growth in pure culture 
in various media, and to ascertain whether it produces 
certain chemical changes in the media by its growth. Its 
ability to grow with or without oxygen, its reaction toward 
staining agents, whether it has independent motion or not, 
and its effects upon animals by inoculation, are also points 
which may have to be determined ' to enable one to make 
a positive diagnosis of the species to which the organism 
belongs. 



2l6 PATHOLOGICAL TECHNIQUE. 

METHODS OF OBTAINING PURE CULTURES. 

When it is desired to obtain a pure culture of bacteria, a 
colony or a portion of a colony of the organism is secured 
on the end of the sterile platinum wire, and transferred by 
this means to the culture-medium in another test-tube. The 
bacteria thus sown in the fresh culture-medium multiply 
there, and produce a growth visible to the naked eye which 
exhibits appearances more or less characteristic of the 
species. This growth, if the medium be a solid one, will 
usually be in the form of confluent colonies ; if the medium 
be a fluid one, the growth may appear as a sediment with or 
without clouding of the liquid, or it may manifest other 
peculiarities according to the species to which the organism 
belongs. If other bacteria are present in the culture from 
which it is desired to obtain material for a pure culture, it is 
important that the material should be taken from a colony 
of the organisms which is well separated from other colonies 
— i. e. that the colony should be a so-called " discrete " one. 

In transplanting, the culture-tube containing the colony 
and the culture-tube that is to be infected from it are held 
side by side in the left hand in a slanting position in such a 
way as to give a good view to the operator of the surface of 
the media in each, while the cotton stoppers are removed 
and held between the fingers of the same hand (Fig. 19). 
The object of holding the tubes in a slanting position is to 
offer less chance of contamination from bacteria gaining en- 
trance to the culture-medium from the air. 

The platinum wire, which is manipulated by the right 
hand, is first sterilized by holding in the Bunsen flame until 
it glows, and then cooled by contact with the media to be 
infected, after which its free end is carefully brought in 
contact with the discrete colony or pure culture-growth, 
and immediately inserted into the sterile tube to inoculate 
it. The manner of inoculating the sterile culture-medium 
in the other tube with the infected platinum wire will vary 
with the form and character of the culture desired. 

If the medium to be inoculated is a fluid one, the wire is 
simply immersed in it and moved back and forth once or 



CULTURE METHODS. 



217 




twice. If the medium be a solid one in the form of a slant, 
the infected end of the wire is drawn over the surface once 
or twice from the bottom of the slant to its upper end ; or 
if the solid medium in the tube be arranged for a stab cul- 
ture (seepage 196), the infected wire is to be plunged once 
through the center of the 
mass to the bottom of the 
tube. After the tubes have 
been inoculated as above in- 
dicated, the wire is to be im- 
mediately withdrawn and 
the cotton stoppers replaced. 
They are then to be placed 
in the incubator for devel- 
opment. Gelatin cultures, 
however, must not be so 
treated, but are to be kept at 
room-temperature, for the 
heat of the incubator would 
cause the gelatin to become 
fluid. 

These details as to the manner of manipulating the cul- 
ture-tubes, cotton stoppers, and platinum wire also apply to 
the procedure described below. 

Method of Isolation of a Bacterium in Pure Cul- 
ture from a Mixed Growth. — If there is a more or less 
confluent growth of colonies of various kinds in a culture- 
tube, and it is desired to isolate a pure culture of one of the 
species of bacteria present, it is obvious that the first step is 
to obtain separate or " discrete " colonies of that organism. 
This is accomplished by securing a minute quantity of the 
growth on the end of the sterilized platinum wire (preferably 
from a spot where the organism is prevalent), and distrib- 
uting this over the surface of a sterile blood-serum tube by 
gently rubbing the end of the infected wire as thoroughly 
as possible over it. The wire is then sterilized in the Bunsen 
flame, cooled in the water of condensation of a second sterile 
blood-serum tube, next touched to the infected surface of the 



FIG. 19 — Method of holding tubes dur- 
ing inoculation. 



218 PATHOLOGICAL TECHNIQUE. 

first tube, and the wire thus infected gently and thoroughly 
rubbed over the surface of the second. In a similar manner 
a third tube is then infected from the second, and then all the 
tubes placed in the incubator for eighteen to twenty-four hours. 
It is evident that comparatively few bacteria will be sown on 
the medium of the second tube, and still fewer on that of the 
third, so that the number of colonies which develop in the 
second tube will be less numerous than in the first tube, 
and those in the third tube still smaller in number. There- 
fore, in either the second or the third tube, or in both, the 
bacteria sown may be sufficiently few for discrete colonies to 
develop from them, and among these there may be some 
composed of the bacterium which it is desired to isolate. 
From one of such discrete colonies pure cultures may then 




Fig. 20 — Diluting cultures. 

be prepared as described above. The second and third 
tubes used in this method are called " dilutions." The 
details of the manner of manipulating the tubes, etc. in this 
method may be understood from the description given on 
page 216 and from Fig. 20. 

The Plate Method of Petri. — Another method for obtaining 
discrete colonies of an organism from a mixed growth of sev- 
eral species is that known as the plate method of Petri. This is 
a modification of the original complicated method of Koch. 

The method consists in making " dilutions " in melted 
agar-agar or gelatin tubes, and then pouring the infected 
medium into shallow glass dishes (Fig. 21) previously steril- 
ized, in which it is allowed to solidify. A few bacteria are 
thus distributed throughout a thin layer of culture-medium 






CULTURE METHODS. 



219 



in the "dilutions," and the colonies which develop from them 
are then more or less separated from one another, so that 
pure cultures may be obtained from them. In carrying out 
this method the procedure is as follows : 

Three sterile gelatin or agar-agar tubes are melted by heat 
and placed in a water-bath warmed to between 40 and 42 ° 
C. for several minutes, to bring the culture-medium to this 
temperature. This temperature is important especially in the 
case of agar-agar, for it is just above the solidifying point of 
that medium (3 8° C.) and yet not injurious to the vitality of 




FIG. 21. — Petri dish with colonies. 

the bacteria. The tubes are then infected successively from 
the bacterial growth or from the pathological material from 
which it is desired to obtain discrete colonies, in the same 
manner as described for the method with blood-serum tubes 
— viz. one tube being inoculated from the growth or tissue, 
a second tube or dilution from the first tube, and a third tube 
or dilution from the second tube, the platinum wire being 
sterilized after each inoculation. For making the " dilu- 
tions " a platinum wire bent into the form of a small loop 
(see page 213) is to be used, and as much of the culture-fluid 
as will adhere to it used for inoculating. The wire should 
be moved back and forth several times in the medium of each 
tube when inoculating it, in order to ensure a good distribu- 
tion of the bacteria throughout the fluid. The contents of 
each tube thus inoculated are then poured into sterilized Petri 



220 PATHOLOGICAL TECHNIQUE. 

dishes, in which the culture-medium solidifies in a thin 
layer. 

The Petri dishes (Fig. 21) are of clear glass, circular in 
form, 10 cm. in diameter, and about 1 cm. deep. Each is 
provided with a loosely fitting flat cover of glass. These 
dishes with their covers are to be sterilized before using by 
placing them in the steam sterilizer for half an hour or by 
heating them to 150 C. in the hot-air sterilizer. When cool 
they are ready to receive the contents of the inoculated test- 
tubes. In pouring, the cover of the dish is not to be removed 
any more than is necessary, and it is to be immediately re- 
placed, so that contamination from the air may be better 
avoided. It is very desirable that there be no dust about the 
place where the dishes are "poured," and no currents of air. 

If agar-agar is used, the dishes thus prepared are to be 
put in the incubator for eighteen to twenty-four hours as 
soon as the medium is solid, which it becomes in a few min- 
utes ; but if gelatin be used, the dishes are to be set aside in 
a cool place, free from dust, to solidify, and are then to be 
kept at room-temperature for several days. Colonies first 
begin to appear in the gelatin usually after forty-eight hours. 

The method of Petri is of great utility in the study of bac- 
teria from the botanical standpoint, for it is especially adapted 
for the study of the appearances of colonies under the low 
power of the microscope. It is, however, inferior to the 
method with blood-serum tubes for routine pathological 
work, for the following reasons : First : Certain pathogenic 
bacteria grow only feebly on the culture-media which it is 
necessary to employ in this method, while they grow com- 
paratively vigorously on blood-serum. Second: The method 
is complicated and much more troublesome and time-con- 
suming than the simple method described above. 

CULTIVATION WITHOUT OXYGEN (ANAEROBIC 
CULTURES). 

Of the numerous methods and modifications of methods 

that have been proposed for the cultivation of anaerobic 

bacteria, only those are given here which have worked sue- 



CULTURE METHODS. 221 

cessfully in our hands, or are regarded as the simplest and 
most practical. 

Culture-media for Anaerobic Bacteria. — The agar- 
agar, gelatin, or bouillon used for the cultivation of ana- 
erobic bacteria should contain I per cent, glucose. These 
media should not be more than two weeks old for the best 
results. 

Their reaction is of the greatest importance, and should 
be adjusted by titration (see page 202). In the case of gel- 
atin and of agar-agar the reaction should be 1 per cent, or 
1.5 per cent, of normal acidity to phenolphthalein. In the 
case of glucose bouillon, however, a more rapid growth 
is obtained with a reaction of less than 1 per cent, normal 
acidity to phenolphthalein. A degree of acidity greater than 
this is probably a frequent cause of failure to obtain cultures 
of obligate anaerobes in bouillon. Therefore, it is of the 
utmost importance, in working with glucose bouillon, to be 
sure that it has the proper reaction at the time of its use. 

The culture-medium must be thoroughly boiled imme- 
diately before inoculation in order to expel absorbed oxygen. 
It is then to be cooled rapidly by immersing the tube in cold 
water, and is to be inoculated within a few minutes after- 
ward. 

Method of Liborius (Fig. 22). — This consists in culti- 
vating the bacteria in the depths of solid media in test-tubes 
filled to a considerable height, so that oxygen cannot pene- 
trate to them through the thick layer of medium. 

A test-tube is filled about three-quarters full x of sterile glu- 
cose gelatin or glucose agar-agar, and its contents boiled for 
a few minutes to expel the excess of oxygen from the me- 
dium. The tube is then immersed in cold water to cool its 
contents rapidly, and then, before the medium becomes solid, 
the tube is placed in a water-bath at 3 8° to 40 C. for a 
few minutes. When the medium may be assumed to have 
reached this temperature, it is inoculated with the material 
from which a growth is sought to be obtained, and then 
rapidly solidified in cold water. The colonies of anaerobic 

1 The tube need not be filled more than half its length. 



222 



PA THOL O GICAL TECHNIQ UE. 



bacteria develop only in the deeper layers of the culture- 
medium. These colonies may be made accessible for sub- 
cultures either by breaking the tube or by removing the 
overlying portions of the culture-media by means of a stout 
platinum wire, previously sterilized in a flame. For taking 
out colonies for transplantation, a capillary glass tube, ster- 
ilized in a flame, may be found useful in place of the pla- 
tinum wire. In inoculating the tube, care should be taken 
to secure a good distribution of the bacteria through the 
medium by manipulating the platinum wire. 



><\ 



Fig. 22. — Liborius's method of 
making anaerobic cultures. 



Fig. 23. — Buchner's method of 
making anaerobic cultures. 



This method will be found very practical for obtaining 
pure cultures from mixed growths if dilutions (see page 217) 
be made. In making dilutions it is well to use a tube of 
bouillon or sterilized water for the first tube, thus econo- 
mizing medium, for the first tube will usually have so many 
colonies that no colonies suitable for sub-cultures will be 
available. 

The microscopical appearances of the colonies may be 
studied by placing thin slices of the medium, containing 
the colonies, on a slide. These slices may be easily ob- 



1 



CULTURE METHODS. 223 

tained with the aid of a stout platinum wire with a flattened 
end, more or less bent. 

Anaerobic bacteria grow readily in " deep slab " cultures. 
In these cultures the medium should fill the tube to almost 
half its height at least. After inoculation some melted 
medium may be poured in so as to fill the tube to an addi- 
tional height of some centimeters, but this is not necessary. 

Simple Anaerobic Plate-cultures. — These are prepared 
like the ordinary Petri plate-cultures (see page 218) except 
that the melted culture-medium is poured into the upturned 
larger dish, or cover, of the pair, while the smaller dish is 
then placed, bottom surface downward, in the melted culture- 
medium, and allowed to settle by its own weight into the 
fluid medium. The dishes are not disturbed until the 
medium has hardened. Sufficient medium should be used 
to fill the space between the sides of the dishes. This quan- 
tity will be about 10 c.c. By slightly inclining the smaller 
dish in placing it in the melted medium, air-spaces can be 
easily avoided. 

By this method the colonies develop in a thin layer of 
culture-medium enclosed between glass surfaces. The 
method gives a good chance to study the microscopical 
characters of the colonies. Surface colonies are, of course,, 
not obtained by this method. The colonies are easily made 
accessible for transplantation by separating the dishes from 
one another. The layer of culture-medium will adhere to 
one dish or the other. 

In order to avoid contamination, the dishes should be 
arranged in the manner above described during their ster- 
ilization previous to using. 

Buchner's Method. — This method consists in cultivating" 
bacteria in an atmosphere from which the oxygen has been 
absorbed by a mixture of alkali and pyrogallic acid. Tube- 
cultures, or cultures in Petri dishes, may be used. They 
should be placed in some form of a glass chamber, which is 
closed air-tight, along with the necessary quantity of alkali 
and pyrogallic acid mixture. In preparing the apparatus, 
the pyrogallic acid (in powder) is placed first in the chamber 
along with the culture tubes or plates, then the necessary 



224 PATHOLOGICAL TECHNIQUE, 

quantity of a solution of potassium hydroxid (i : 10) is run 
in, and the chamber quickly closed. For single tube-cul- 
tures a large test-tube provided with a tightly fitting rubber 
stopper, which is sealed in position with wax, may be used 
for the air-tight chamber (see Fig. 23). The culture-tube is 
to be elevated above the surface of the reducing mixture by 
means of a bent wire. 

If a number of tube-cultures or Petri plate-cultures are 
desired, the glass chambers known as Novy's jars are very 
satisfactory to use. The joints of this apparatus should be 
well smeared with vaselin. To avoid breakage the test- 
tube containing the inoculated culture-medium may be held 
in a beaker, with some cotton at the bottom, while in the 
apparatus. Petri plate-cultures may be placed one above 
another in the jar, the bottom plate being supported above 
the level of the reducing fluid by some sort of wire frame. 

It is necessary to seal up the apparatus quickly in order 
to obtain the full benefit of the oxygen-absorbing power 
of the pyrogallic acid. The quantity of pyrogallic acid em- 
ployed should be about 1 gram for each 100 c.c. of air-space 
to be exhausted of oxygen, and for every gram of pyro- 
gallic acid 10 c.c. of the solution of potassium hydroxid 
should be used. 

Hans Zinsser's Method for Anaerobic Plate- cultures. — 
The method is described by Dr. Zinsser as follows : 

" The apparatus used consists of two circular glass dishes, 
fitting one into the other, as do the halves of a Petri dish, and 
similar to these in every respect except that they are higher, 
and that a slightly greater space is left between their sides 
when they are placed together. The dishes should be about 
f to 1 inch in height ; they need be of no particular diameter, 
although those of about the same size as the usual Petri 
dishes are most convenient. The sole requirement neces- 
sary for successful plating is that the trough left between 
the two plates when put together shall not be too broad, a 
quarter of an inch being most favorable. 

" Into the smaller of these plates the inoculated agar is 
poured, exactly as is done into a Petri dish in the ordinary 



CULTURE METHODS. 225 

aerobic work. Prolonged boiling of the agar before plating 
is not essential. When the agar-film has become sufficiently 
hard on the bottom of the smaller dish, the entire apparatus 
is inverted. The smaller dish is now lifted out of the larger, 
and placed, still inverted, over a moist surface — a towel or 
the wet surface of the table — to prevent contamination. Into 
the bottom of the larger dish, which now stands open, there 
is placed a quantity (i to 2 drachms) of dry pyrogallic acid. 
Into this, over the pyrogallic acid, the smaller dish, still in- 
verted, is then placed. A strong solution of sodium hydrate 
is poured into the space left between the sides of the two 
dishes, in quantity sufficient to fill the receiving one half full. 
While this is gradually dissolving the pyrogallic acid (and 
this is the only step which requires speed), albolene, or any 
other oil, is dropped from a pipette, previously filled and 
placed in readiness, into the same space, thus completely 
sealing the chamber formed by the two dishes. 

" If these steps have been performed successfully, the 
pyrogallic solution will at this time appear of a light brown 
color, and the smaller plate, with its agar-film, will float un- 
steadily above the other. Very rapidly, as the pyrogallic 
acid absorbs the free oxygen in the chamber, this plate is 
drawn down close to the other, and the acid assumes .a 
darker hue, which remains without further deepening even 
after three or four days' incubation." 

Wright's Method. — The method depends upon the ab- 
sorption of oxygen by an alkaline solution of pyrogallic 
acid, as in the well-known method of Buchner. It is appli- 
cable to culture in test-tubes and in flasks. The details of 
the method are as follows : 

After the culture-medium in the test-tube has been inocu- 
lated, the cotton stopper is thrust sufficiently far down into 
the test-tube so that the upper end of the cotton stopper 
lies about 1 5 mm. below the mouth of the test-tube. It is 
usually desirable to cut off a part of the protruding portion 
of the cotton before doing this. Now fill the space in the 
tube above the cotton stopper with dry pyrogallic acid. 
Next pour quickly onto this pyrogallic acid enough of a 

15 



226 



PA THOL O GICA L TE CHNIQ UE. 



strong watery solution of sodium hydrate to dissolve it all; 

avoid pouring on an excess ; for a test-tube f of an inch in 

diameter about 2 c.c. will be an ample quantity. Then, as 

quickly as possible, insert firmly a rubber stopper in the 

mouth of the tube so as to close it tightly. The culture is 

then ready to be set aside for development. 

The cotton of the stopper should be of a kind that will 

readily absorb fluids. 

The solution of sodium hydrate consists of one part of 

sodium hydrate in sticks and two parts of water. 

It may be thought that there is danger of contaminating 

the culture-medium from the alkaline pyrogallic acid mix- 
ture running down the sides of the 
tube. This does not occur, be- 
cause the mass of the cotton stop- 
per is sufficiently large to absorb 
completely the quantity of fluid in 
it, with a good margin to spare. 

This simple method has given 
satisfactory cultures of the tetanus 
bacillus obtained from cases of 
tetanus in the Massachusetts Gen- 
eral Hospital and of other obligate 
anaerobic bacteria. It can be ap- 
plied to all forms of test-tube cul- 
tures, both in solid and fluid media, 
including Esmarch roll-cultures. 
In applying the method to Esmarch 
roll-cultures the mixture of pyro- 
gallic acid and alkali should be 
placed in the cotton, and the rub- 
ber stopper inserted before the tube 
is rolled on the ice. Glucose-agar 
readily lends itself to Esmarch roll- 
cultures if the tubes are kept in a 
slanting position during growth. 

The accompanying photograph 
shows the appearance of a bouillon 

tube prepared according to this method (Fig. 24). 




Fig. 24. — Wright's method 
for the cultivation of anaer- 
obes. 



CULTURE METHODS. 



227 



The Determination of the Motility of Bacteria. — 

This is done by observing the individual organisms, un- 
stained, in a drop of bouillon or similar fluid under the oil- 
immersion lens. For this purpose a so-called " hanging drop" 
is prepared, for which a special form of slide known as a 
"hollow slide" is necessary. The hollow slide is a slide 
having a shallow- circular concavity, about 1 cm. in diam- 
eter, ground out in its center (Fig. 25). 

In preparing" a hanging drop the procedure is as follows : 
A small drop of a bouillon culture or of the water of con- 
densation of a blood-serum or agar-agar slant is placed in 
the center of a cover-glass by means of the platinum wire t 
The cover-glass is then placed, drop downward, over the 
circular depression in the hollow slide. To hold the cover- 
glass in its place and to prevent evaporation of the fluid in 
which the organisms are suspended, a little vaselin is painted 
around the margin of the depression before placing the 
cover-glass in position. The hanging drop thus prepared is 




Fig. 25. — The " hanging drop " seen from above and in profile. 

then examined by focusing upon it with the oil-immersion 
lens, a small aperture of the iris diaphragm of the conden- 
ser being used to render the bacteria visible by refraction. 
To facilitate focusing, the edge of the drop should b$ 
brought into the center of the field of the low-power ob- 
jective, and then the oil-immersion put in place and focused 
upon it, the edge of the drop being more readily seen as a 
sharp line, owing to refraction, than the organisms. Great 
care is necessary to avoid breaking the cover-glass in the 
effort to bring the bacteria into view. Hanging drops may 



228 PATHOLOGICAL TECHNIQUE. 

also be prepared from suspensions of bacteria grown on solid 
media, by mixing a portion of the growth with a small quan- 
tity of bouillon. 

In the study of spore-formation the hanging drop is of great 
utility. Here the slide and cover-glass must be carefully steril- 
ized before using, the cavity between the cover-glass and the slide 
well sealed with vaselin, and other precautions taken to prevent 
contamination of the drop with other bacteria. The preparations 
may be placed in the incubator or on a "warm stage " and the 
process of spore-formation followed. 

H. W. Hill's "Hanging-block" Method for the Observa- 
tion of Developing" Bacteria. — " Pour melted nutrient agar 
into a Petri dish to the depth of about one-eighth to one-quarter 
inch. Cool this agar and cut from it a block about one-quarter 
inch to one-third inch square, and of the thickness of the agar 
layer in the dish. This block has a smooth upper and under sur- 
face. Place it, under surface down, on a slide, and protect it 
from dust. Prepare an emulsion in sterile water of the organism 
to be examined if it has been grown on a solid medium or use a 
broth culture ; spread the emulsion or broth upon the upper sur- 
face of the block as if making an ordinary cover- slip preparation. 
Place the slide and block in a 37 C. incubator for five or ten 
minutes to dry slightly. Then lay a clean sterile cover-slip on 
the inoculated surface of the block in close contact with it, usu- 
ally avoiding air-bubbles. Remove the slide from the lower sur- 
face of the block, and invert the cover-slip so that the agar block 
is uppermost. With a platinum loop run a drop or two of melted 
agar along each side of the agar block, to fill the angles between the 
sides of the block and the cover-slip. This seal hardens at once, 
preventing slipping of the block. Place the preparation in the in- 
cubator again for five or ten minutes to dry the agar seal. Invert 
this preparation over a moist chamber and seal the cover-slip in 
place with white wax or paraffin. Vaselin softens too readily at 37 
C. , allowing shifting of the cover-slip. The preparation may then 
be examined at leisure. For bacillus diphtherias and organisms 
of similar size, Zeiss ocular 5, objective y 1 ^, oil immersion, and a 
Welsbach light prove satisfactory, although a lower ocular and 
higher objective are better. The Abbe condenser is not used. If 
preferred, the Welsbach light may be concentrated by a four-inch 
lens, focal length seven inches. An incandescent electric lamp 
is very difficult to focus and does not yield good results." . . . 

"Bacteria multiplying readily at room- temperature can be ob- 
served in such a preparation exactly as an ordinary hanging drop 
is observed, except that the slide should be secured rigidly in 
some way to the microscopic stage to prevent shifting. For 
bacteria growing best at 37 C. a warm stage is required." 






CULTURE METHODS. 2 29 

THE INOCULATION OF ANIMALS. 

The animals ordinarily used in the laboratory are guinea- 
pigs, rabbits, and mice. The instruments, etc. used in the 
inoculation of animals should be sterilized beforehand, but 
strict surgical asepsis is not necessary as a rule. 

Guinea-pig's are in most instances inoculated either sub- 
cutaneously or into the peritoneum. 

Subcutaneous inoculation is effected either by injection with 
a hypodermic syringe or by the introduction of the material 
to be inoculated through a small incision in the skin. The 
best point for subcutaneous inoculation is the tissue of the 
anterior abdominal wall. 

In inoculating, the animal is to be held abdomen upper- 
most by an assistant, who grasps the neck and fore quarters 
with one hand and the hind quarters with the other. If the 
skin is to be incised, the hair about the point of inoculation 
is to be cut short with a pair of scissors and the skin cleansed 
with soap and water. An incision is then to be made about 
8 or 10 mm. long through the skin, including the subcuta- 
neous tissue, and the superficial tissues separated from the 
muscle for a distance of 10 or 15 mm. toward one side of 
the wound by inserting the points of scissors or other in- 
strument, so as to form a " pocket " beneath the skin. In 
this "pocket" the material for inoculation is introduced, 
either on the platinum wire (see page 213) or by means of 
small forceps. 

If pieces of tissue are used, it may be well in some cases 
to close the wound by one or two sutures in order to prevent 
the extrusion of the material after the release of the animal. 

Intraperitoneal inoculation may be performed essentially as 
above indicated. If the inoculation be by incision, the open- 
ing into the peritoneal cavity should be as small as possible, 
and the wound should be firmly closed with silk sutures in 
order to prevent extrusion of the intestines. 

In inoculating with the hypodermic syringe the needle 
should not be pushed in too far or the intestines may be 
wounded. The needle is best introduced a little to one side 
o£ or slightly below, the umbilicus. 



23O PATHOLOGICAL TECHNIQUE. 

Rabbits. — These animals may be inoculated both sub- 
cutaneously and intraperitoneal^, essentially as described for 
guinea-pigs. 

In lifting or in carrying rabbits from one place to another 
the animals are to be grasped by the ears. During the ope- 
ration of inoculating, the assistant grasps the ears with one 
hand and the hind legs with the other, while the body of the 
animal rests upon the table, abdomen uppermost. Rabbits 
held for a few seconds in this position usually become per- 
fectly quiet, and often do not show any evidence of pain 
during the operation. 

Intravenous inoculation is usually done on rabbits, because 
of the ease with which the needle of a hypodermic syringe 
may be introduced into the long and prominent marginal 
vein of the ear. In inoculating in this manner the tip of the 
ear is held by the thumb and fingers of the left hand, while 
the right manipulates the syringe, the needle of which is 
pushed through the skin of the external surface of the ear 
into the vein which runs along the outer margin of the ear 
(Fig. 26). 

By the exercise of care and gentleness the animal may be 
thus inoculated without being held by an assistant, especially 
if the fur between the ears be stroked for a short time just 
before the introduction of the needle. In some cases it may 
be necessary to anesthetize the. animal on account of violent 
struggling. (See below.) 

Injection of bacteria into the mesenteric veins by means of the 
hypodermic syringe, after laparotomy, may be performed both 
on rabbits and on guinea-pigs. This is to be done under anes- 
thesia. Ether is very satisfactory for this purpose. Guinea-pigs 
bear it well, but it is to be used with caution on rabbits. With the 
latter animals death is liable to occur if the ether is " pushed ' ' after 
complete anesthesia is established. Rabbits once thoroughly anes- 
thetized seem to remain so for a considerable time without addi- 
tional ether being necessary. The incision for this form of inocu- 
lation should be in the lower half of the abdominal wall in the 
median line, for in this region the coils of the small intestine are 
most numerous. The length of the incision should be about 2 
cm. Several loops of intestine are brought out through the 
wound, and a mesenteric vein, of the proper size to admit the 
needle of a hypodermic syringe, is sought for. When found the 
needle is to be introduced and held firmly in position while an 



CULTURE METHODS. 23 1 

assistant carefully presses inward the piston of the syringe. After 
the injection of the material the needle is withdrawn, the punc- 
tured vein picked up with the artery- forceps, and the vessel tied 
on both sides of the puncture with silk thread. The loops of the 
intestine are then replaced and the wound closed in two layers, 
one consisting of the muscles and peritoneum, the other of the 
skin. The so-called " button-hole stitch" with silk thread is 
very well fitted for the closing of the wound. 

Little or no aseptic precautions are necessary to obtain primary 
union in the wound. Before the operation, however, the hair of 
the region should be cut off close and the skin cleansed with soap 
and water. 

This form of inoculation may be useful in studying the local 
effects of bacteria upon liver-tissue, for large numbers of them 




Pig. 26. — Method of making an intravenous injection into a rabbit. Observe 
that the needle enters the posterior vein from the hairy surface (McFarland). 

will be lodged in the capillaries of the liver, and microscopical 
sections of any part of the organ will contain them, so that any 
local lesion produced by them may be subjected to observation 
after variable intervals of time. 

Mice are usually inoculated subcutaneously at the root 
of the tail. The animal, manipulated by means of chemists' 
crucible tongs or a similar instrument grasping his tail, is to 
be persuaded to crawl into a cylinder of wire gauze, about 
8 to 10 cm. long and about 3 cm. in diameter, which is fixed 
on a small board. The cylinder is open at both ends, and 
when the mouse has crawled into it — a thing which he will 



232 



PA THOL O GICAL TE CHNIQ UE. 



readily do — the end near his tail is bent inward so as to pre- 
vent him from backing out of it, while an ordinary small 
screw-clamp is adjusted firmly to his tail to prevent his es- 
caping through the other end. The animal is thus secured 
and ready for the operation of inoculation. A more com- 
plete form of this apparatus, with a fixed clamp for the ani- 
mal's tail, is shown in Fig. 27. 

In making the inoculation the mouse is pulled backward 
by the tail until his rump is exposed in the end of the cylin- 
der, and then with small scissors the hair is cut away over a 
space, approximately 1 cm. square, about the root of the 
tail. In the center of this a small opening is made through 
the skin 3 or 4 mm. long with small scissors, and through 
the opening the points of the scissors are passed anteriorly 
beneath the skin for a distance of about 1 cm., so as to make 
a " pocket " or cavity by separating the skin from the mus- 
cles. Into the cavity thus formed the material for inocula- 
tion is then to be introduced by means of the platinum wire. 
As a rule, white mice are to be preferred to the wild brown 
variety, on account of the greater ease with which they may 
be handled. 




Fig. 27. — Mouse-holder, with mouse in position for inoculation. 

Mice may also be inoculated in the peritoneal cavity by intro- 
ducing a very few drops of a suspension or a bouillon culture 
of an organism with a hypodermic syringe. 



The quantity of bacteria used for purposes of inoculation 
varies with the organism and with the end in view. In gen- 
eral, it may be said that in inoculating with the growth from 



CULTURE METHODS. 233 

a solid medium with the platinum wire one or two loopsful 
are used. If bouillon cultures are employed, the quantity 
injected varies from -^ c.c. to 1 c.c. in most cases. 

In cases where a " suspension " of the growth on a solid 
medium is injected the same quantities are used as in the 
case of bouillon cultures, the density of the suspension de- 
pending upon the operator. A " suspension " may be con- 
veniently prepared by pouring 5 or 8 c.c. of sterile bouillon, 
sterilized water, or 0.6 per cent, sodium chlorid solution (ster- 
ilized) into the tube containing the growth upon solid medium, 
then breaking up the colonies of the growth with the plat- 
inum wire, and shaking the tube. 

The Care of Animals. — Inoculated guinea-pigs should 
be kept in boxes or cages so arranged as to permit of clean- 
ing and disinfection. Cages made of a combination of gal- 
vanized-iron wire netting and galvanized sheet iron are 
to be preferred. The bottom of the cage should contain 
sawdust, and the top may be made to open on hinges. Good 
dimensions for such cages are 16 inches long, 10 inches wide, 
and 10 inches high. They may be satisfactorily disinfected, 
in most instances at least, by washing with boiling water. 

Inoculated mice are well kept in large glass jars with per- 
forated covers. A small amount of tissue paper should be 
provided for bedding. 

The "stock" guinea-pigs and rabbits may be kept together 
in a pen which should have light and ventilation. Guinea- 
pigs breed readily and their young thrive, but this is not 
usually the case with rabbits. Mice may be kept for use in 
a woven-wire cage set in a sheet-iron pan, which will permit 
of the easy removal of excreta. Some raw cotton should be 
furnished for bedding. The young of white mice are diffi- 
cult to raise to maturity. 

Food. — Rabbits and guinea-pigs eat the same things. In 
summer-time, grass, green corn-husks, and green vegetables 
generally are good food for them. In winter, carrots and oats 
form a satisfactory diet. Fresh water should also be supplied. 

Mice may be fed on stale bread soaked in water, oats, bird- 
seed, and occasionally some cheese. Fresh water should be 
furnished, and, if possible, a little milk sometimes. 



METHODS OF STAINING BACTERIA. 



THE STAINING OF BACTERIA IN SMEAR PREPARA= 

TIONS. 

A cover-glass preparation is made as follows: A very 
small amount of tissue or material to be examined is thinly 
spread over the surface of a clean thin cover-glass with the 
platinum wire or "loop" described on page 213 so as to give 
a streaked appearance to the surface, but not a definite 
layer, which is ordinarily too thick for satisfactory exami- 
nation. The cover-glasses are best kept in alcohol, and as 
required for use wiped dry with a soft cotton cloth. 

For preparations from cultures a minute proportion of 
a colony or bacterial growth, the component organisms of 
which are to be examined, is picked up on the end of the 
platinum wire, which has been previously heated in the 
Bunsen flame and cooled, and is thinly distributed on the 
surface of a cover-glass by gentle movements of the plati- 
num wire. It is very important that the bacteria should 
be more or less separated from one another in places, so that 
a good view of the individual organisms may be obtained. 
This can often best be effected by placing a minute drop 
of water on the cover-glass first, and then moving the 
infected end of the platinum wire back and forth through 
this. 

The charged cover-glass is then dried by holding it in 
the fingers over the flame of a Bunsen burner, and when 
dry it is placed, charged surface uppermost, in the grasp 
of a pair of cover-glass forceps, 1 by means of which it is 
passed rapidly three times through the flame of a Bunsen 
burner or alcohol lamp. This "fixes" the material on the 

1 The form of cover-glass forceps known as Stewart's is recommended. 
It may be obtained from American dealers in bacteriological apparatus. 
234 



METHODS OF STAINING BACTERIA. 2$$ 

glass, and the preparation is then ready for staining by one 
of the various methods given below. In staining, the 
cover-glass is held by means of the forceps with the charged 
side uppermost and level, and the surface is then com- 
pletely covered with the staining fluid, which is poured 
upon it from a dropping-bottle. 1 It may then be heated 
over the flame of the Bunsen burner, washed in water, and 
submitted to any further manipulation which may be neces- 
sary while still in the grasp of the forceps. When the 
staining is completed the preparation is next to be pre- 
pared for microscopic examination. This is done by plac- 
ing the cover-glass, with as much water as will adhere to 
it, charged side downward, on a "slide," and then removing 
all remaining water, except a thin film of water between the 
slide and cover-glass, by gentle pressure with several thick- 
nesses of filter-paper. The preparation is then ready for 
examination with an oil-immersion lens. The presence of 
this film of water is very essential for a satisfactory exam- 
ination with the microscope, and its evaporation may be 
compensated for by a drop of water placed at the edge of 
the cover-glass. The preparation may also be mounted in 
balsam after carefully drying it first between filter-paper 
and then holding it in the fingers over the Bunsen flame, 
but the examination in water mount is better, because the 
apparent size of the bacteria is greater in this than when 
mounted in balsam. 

Simple staining is used for the demonstration of bac- 
teria- in general, and is also useful in gaining an idea of the 
character of the cellular elements in the preparation. 

The staining solutions ordinarily employed are carbol- 
fuchsin, aniline-methyl-violet, and Loffler's alkaline meth- 
ylene-blue. 

Loffler's alkaline methylene-blue solution is perhaps the 
best staining fluid to use for simple staining, for it does not 
stain so diffusely and intensely as do the other commonly 
used dyes, such as fuchsin and methyl-violet, which may 
also be employed. 

1 The form of drop-bottle known by dealers in bacteriological supplies as 
the "T. K. patent," with flat stopper, is the best. 



236 PATHOLOGICAL TECHNIQUE. 

The cover-glass, covered with the staining fluid, should be 
warmed over the Bunsen flame, so that the fluid steams, for 
about fifteen seconds. Boiling should be avoided. The 
preparation is then washed in water for two or three seconds 
and mounted. 

Pappenheim's pyronin and methyl-green mixture may 
be used as above described in place of Loffler's methylene- 
blue solution. This mixture is composed of: 

Saturated aqueous solution of methyl-green, 3-4 parts ; 

" pyronin, 1-1} " 

Bacteria are stained brilliant red and the nuclei of cells are 
stained blue or purple. The mixture is said to keep several 
weeks. 

Gram's Method of Staining". — 1. Cover the preparation 
with aniline-methyl-violet solution for thirty seconds. 

2. Wash in water for two or three seconds. 

3. Cover the preparation with Gram's solution of iodin 
for thirty seconds. 

4. Wash with 95 per cent, alcohol until the color ceases 
to come out of the preparation. 

5. Wash in water for two or three seconds and mount. 
Certain bacteria are stained by this method, while others 

are not. Bacteria when stained by it appear dark blue or 
black, while the nuclei of the cells are rather faintly stained 
or not stained at all. The method is especially useful in the 
demonstration of bacteria which are stained by it when 
they are present in small numbers or when a few Gram- 
staining bacteria are mixed among numbers of bacteria 
which do not stain by this method. It also has some value 
as a means of differentiating between bacteria which may be 
very much alike in size and shape. 

In this connection it should be pointed out that bacteria 
which are stained by this method, when taken from cultures 
a few days old, may not be stained by it if taken from older 
cultures Therefore, Gram's method, if used as a means of 
differentiation, should be applied only to bacteria in actively 
growing cultures. 



METHODS OF STAINING BACTERIA. 



237 



In the following table the behavior of the more important 
pathogenic bacteria toward the method of Gram is indicated : 



Stained by Gram's Method. 
Staphylococcus pyogenes aureus. 
Staphylococcus pyogenes albus. 
Streptococcus pyogenes. 
Pneumococcus. Streptococcus cap- 

sulatus. 
Bacillus scarlatinae. 
Micrococcus tetragenus. 
Bacillus diphtheriae. 
Bacillus tuberculosis. 
Bacillus of anthrax. 
Bacillus of tetanus. 
Bacillus aerogenes capsulatus. 
Bacillus of malignant edema. 



Decolorized by Gram's Method. 
Gonococcus. 

Diplococcus intracellularis meningi- 
tidis. 
Typhoid bacillus. 
Bacillus coli communis. 
Spirillum of Asiatic cholera. 
Bacillus pyocyaneus. 
Bacillus of influenza. 
Bacillus of glanders. 
Bacillus proteus. 
Bacillus mucosus capsulatus. 
Bacillus of dysentery. 
Bacillus of bubonic plague. 
Bacillus of chancroid. 



W. H. Smith's Method of Staining- Bacteria in Sputum. — 
This has been found particularly useful in demonstrating the 
pneumococcus in the sputum. The sputum or other material 
should be fresh. The cover-glasses should be spread as 
thinly as possible and fixed by passing three times through 
the flame in the usual manner. 

1. Stain in aniline-gentian-violet solution for a few 
seconds, gently warming until the staining fluid steams. 

2. Wash in water. 

3. Cover with Gram's solution of iodin for thirty seconds. 

4. Wash with 95 per cent, alcohol until the color ceases 
to come out. 

5. Wash in absolute alcohol for a few seconds. 

6. Stain one to two minutes in a saturated aqueous solu- 
tion of eosin. 

7. Wash with absolute alcohol for a few seconds. 

8. Clear with xylol. 

9. Mount in balsam. 

The pneumococcus is stained blue-black, while the capsule 
is stained pink. With the following modification it has 
been used by Smith as a routine stain for sputum. The 
advantage of this modification is that influenza bacilli and 
other bacteria which do not stain by Gram's method are 
clearly brought out, as are also eosinophilic leucocytes. 



238 PATHOLOGICAL TECHNIQUE. 

This modification consists in washing the preparation with 
Loffler's alkaline methylene-blue solution just after it has 
been stained with eosin, as described above, and then, after 
the excess of eosin has been removed by the methylene- 
blue, steaming the methylene-blue solution for a few sec- 
onds while on the cover-glass. The preparation is then 
washed in water, rinsed with alcohol, cleared with xylol, 
and mounted in balsam. 

The Staining of Spores. — Spores take up the aniline 
dyes with difficulty, probably owing to their dense pro- 
tective envelope. When once stained, however, they do 
not give up their color easily, and resist decolorizing agents. 
The cover-glass preparations should be thinly spread. 

Abbott's Method. — 1. Stain the cover-glass preparation 
deeply with methylene-blue, heating repeatedly until the 
staining solution boils, but do not boil continuously, during 
about one minute. 

2. Wash in water. 

3. Wash in 95 per cent, alcohol containing 0.2 to 0.3 per 
cent, hydrochloric acid. 

4. Wash in water. 

5. Stain for eight to ten seconds in aniline-fuchsin solution. 

6. Wash in water and mount. 

The spores are stained blue and the bodies of the bacteria 
red. 

Moeller's Method. — I. Wash the cover-glass preparation 
in chloroform for two minutes. 

2. Wash in water. 

3. Treat with 5 per cent, solution of chromic acid one- 
half to two minutes. 

4. Wash in water. 

5. Stain with carbol-fuchsin, heating slowly until the 
fluid boils. 

6. Decolorize well in a 5 per cent, solution of sulphuric 
acid. 

7. Wash in water. 

8. Stain in aqueous solution of methylene-blue (1 gram 
to 100 c.c.) thirty seconds. The spores will be red, the 
bodies of the bacteria blue. 



METHODS OF STAINING BACTERIA. 239 

The preliminary treatment with chloroform is to cleanse 
the preparation. 

Fiocca suggests the following rapid method: "About 
20 c.c. of a 10 per cent, solution of ammonia are poured 
into a watch-glass, and ten to twenty drops of a saturated 
aqueous solution of gentian-violet, fuchsin, methylene-blue, 
or safranin added. The solution is warmed until vapor 
begins to rise, then is ready for use. A very thinly spread 
cover-glass, carefully dried and fixed, is immersed for three 
to five minutes (sometimes ten to twenty minutes), washed 
in water, washed momentarily in a 20 per cent, solution 
of nitric or sulphuric acid, washed again in water, then 
counterstained with a watery solution of vesuvin, chrys- 
oidin, methylene-blue, malachite-green, or safranin, accord- 
ing to the color of the preceding stain. This whole process 
is said to take only from eight to ten minutes, and to give 
remarkably clear and beautiful pictures." 

The Staining of Flagella. — All motile bacteria are 
provided with delicate wavy, hair-like prolongations of their 
protoplasm, called flagella, which are of comparatively great 
length. These flagella are the locomotor organs of the 
organism. The number of them attached to each individual 
varies to a considerable extent with the species of the bac- 
teria. Thus the individuals of some species have but one 
flagellum, while the individuals of other species may have 
few or many springing from all parts of the organism. 

The flagella are not rendered visible by the ordinary 
methods of staining, but special methods are necessary for their 
demonstration. These methods depend essentially upon the 
use of a mordant, which causes the flagella to take up the stain. 

The cover-glasses must be absolutely free from grease in 
these methods, so that the watery fluids may be spread 
evenly over them and not run into patches. The cover- 
glasses may be prepared by warming them in concentrated 
sulphuric acid for a time, washing them in water, and keep- 
ing them in a mixture of equal parts of alcohol and strong 
ammonium hydroxid solution. 

When used they are to be dried on a cloth which has pre- 
viously been soaked in ether and allowed to dry, in order that 



24O PATHOLOGICAL TECHNIQUE. 

it may contain no trace of fat. Another way to treat the 
cover-glasses is to take them from alcohol, dry them with a 
clean cloth, and then heat them by means of the cover-glass 
forceps in the Bunsen flame to burn off any fat or grease. 

The bacteria must be distributed upon the cover-glass well 
separated from one another in these methods. They should 
not be subjected to too much manipulation in doing this, for 
the flagella are readily broken off. A good way is to make 
a dilute suspension of the bacteria in distilled water, and 
place one or two loopfuls of this on the cover-glass, not 
spreading with the loop, but making the suspension flow 
over the surface by inclining the cover-glass. 

Another way is to place two drops of water on a cover- 
glass — to draw the infected wire once through one of them 
across the surface, and then once through the other drop, 
thus making two streaks. This subjects the bacteria to less 
manipulation and gives a good distribution in places. 

The cover-glasses prepared as above indicated are to be 
allowed to dry in the air, and are then to be heated for a 
few seconds over a flame while held between the fingers. 
They are then ready to be stained by any of the methods 
given below. The cultures used for the preparations should 
not be older than eighteen to twenty-four hours. Solid 
culture-media, such as agar-agar, should be employed. 

Loffler's Method. — Treat the preparation for about one 
minute with the freshly filtered mordant solution, which is — 

Aqueous solution of tannic acid (20 grams tannic acid 

to 100 c.c. water), ioc.c. ; 

Cold saturated solution of ferrous sulphate, 5 c.c. ; 

Saturated aqueous or alcoholic solution of gentian- 
violet or fuchsin, 1 c.c. 

The cover-glass is to be covered with this while held with 
the cover-glass forceps, as in ordinary methods of staining. 
The mordant, thus placed on the cover-glass, may be gently 
heated by holding the preparation high over the flame for a 
period of about one minute, but it must not be boiled. After 
this the preparation is to be washed in water, and then 
stained with a freshly prepared and filtered solution of ani- 



METHODS OF STAINING BACTERIA. 24 1 

line-gentian-velvet or aniline-fuchsin, with gentle heating for 
thirty to sixty seconds. It is then again washed in water, 
and mounted in water or balsam for examination. 

In using this method, as well as others, an important thing 
to avoid is overheating. The mordant may be freshly mixed 
every time or kept indefinitely for use. 

The ferrous sulphate solution should always be freshly 
prepared, for it rapidly decomposes. The solution of tannic 
acid keeps well, however. 

The addition of varying quantities of acids or alkalies for 
different species of bacteria, as recommended by Loffler, is 
not necessary. 

Williams' Method. — This is a modification of van Er- 
mengem's method along the lines of the modification of 
Hinterberger and others. It has been adopted by Dr. 
Hugh Williams after a large experience with various 
methods in the Laboratory of the Massachusetts General 
Hospital. 

The method is capable of giving black bacteria and fla- 
gella, with little or no precipitate. The method is as follows: 

1. Cover the cover-glass with a mordant consisting of 

Alumnol, 1 1 per cent, solution, 5 c.c. ; 

Osmic acid, 2 per cent, solution, 5 c.c. ; 

Tannin, 20 per cent, solution, 1 5 c.c. 

Shake the mixture, and add three drops of glacial acetic 
acid, and again shake. 

2. Apply the mordant less than one minute without heat- 
ing. Wash thoroughly in water. 

3. Cover the preparation, during about one minute, with a I 
per cent, solution of silver nitrate to which sufficient ammonium 
hydroxid has been added to keep the silver in solution. 

4. Wash in water. 

5. Wash with 0.6 per cent, solution of sodium chlorid. 

6. Flood the preparation with a 30 per cent, solution of 
ammonium hydroxid, and immediately wash in water. 

7. Apply a few drops of Ortol photographic developer. 

1 Farbwerke vorm. Meister Lucius u. Briining, Hochst a. M., Germany. 
16 



242 PATHOLOGICAL TECHNIQUE. 

The directions for making up this developer come with the 
Ortol. 

8. Wash in water. 

9. Cover with a 1 per cent, solution of gold chlorid 
during a few seconds. 

10. Wash in water, and apply Ortol developer for a few 
seconds. 

11. Wash in water, and cover with a 1 per cent, solution 
of mercuric chlorid for a few seconds. 

12. Wash in water. 

13. Apply Ortol developer for a few seconds. 

14. Wash in water, and repeat the application of chlorid 
of gold, the washing, and the application of the developer 
two or more times. Between the various applications of 
the chlorid of gold the preparation should be inspected 
with a high, dry lens to determine the progress of the stain- 
ing. This is readily done by placing the cover-glass, charged 
side upward, on a slide. In this way the process of impreg- 
nation with gold may be controlled ; for the flagella, if 
stained, may be easily seen with the high-power dry lens. 

The preparation is very conveniently held during the 
process in cover-glass forceps. The washing is best done in 
a small stream of water from a faucet. The various solutions 
are conveniently applied from dropping-bottles, see p. 235. 

It will be seen that the process consists essentially in 
the impregnation of the flagella with silver, followed by 
intensification, in the photographic sense, with mercury and 
gold. The object of the application of the sodium chlorid 
and ammonia is to remove the excess of silver compounds 
which adhere to the surface of the cover-glass in spite of 
washing. This excess of silver compounds is chiefly respon- 
sible for the precipitates which appear on the preparation 
after the intensification. In spite of the application of the 
sodium chlorid and ammonia solutions, some precipitate 
will occur if the intensification is pushed too far. On this 
account it is advisable to observe the progress of the inten- 
sification under the microscope as above indicated. 

Although this method may appear complicated, in practice 
it requires but a few minutes to stain a preparation. 



METHODS OF STAINING BACTERIA. 243 

Claudius' Method for Staining Flagella. — 1. Fix the 
cover-glass or slide preparation by heating in a drying 
oven or hot air sterilizer to no° C. in such a manner that 
this temperature is reached slowly in the course of half 
an hour. During the heating the preparation is to be 
enclosed in dry filter-paper. 






-Typhoid bacilli showing flagella, after Claudius' method; X 1500 (Wright 
and Brown). 

2. Cover the preparation with, or immerse it in, the 
following mixture for twenty to thirty minutes at room 
temperature : 

I per cent, aqueous solution of chromic 

acid, 20 c.c. ; 

Freshly prepared aniline water, 0.5 

Glacial acetic acid, 10 drops. 

The aniline water is prepared by shaking together for a 
few minutes 1 part of aniline oil and 25 parts of distilled 
water and filtering through wet filter-paper. 

3. Wash in water and dry in air. 

4. Mount in balsam or cedar oil and examine with strong 
light and open condenser. 

The method depends on the formation of aniline black in 
the mixture and the deposit of a bluish film on the prepara- 
tion in which the flagella stand out uncolored. 



244 PATHOLOGICAL TECHNIQUE. 

Any film formed in the uncharged surface of the prepara- 
tion may be removed with a towel wet with aniline oil. 

Zettnow's Flagella Staining- Method. — Solution I : Dis- 
solve 2 grams of tartar emetic in 40 c.c. of water. Solu- 
tion II: Dissolve 10 grams of tannin in 200 c.c. of water. 
To the 200 c.c. of Solution II, warmed to 50 or 6o° C, add 
30 c.c. of the tartar emetic solution. The turbidity of the 
mordant should entirely clear up on heating. The mordant 
should keep for months if a small crystal of thymol is added 
to it. 

Next dissolve 1 gram of silver sulphate in 250 c.c. of 
distilled water. Of this solution take 50 c.c. and add to 
it drop by drop ethylamine (this comes in a 33 per cent, 
solution) until the yellowish-brown precipitate which forms 
at first is entirely dissolved and the fluid is entirely clear. 
It requires only a few drops. The bacterial preparations 
prepared as described above are floated in a little mordant 
contained in a Petri dish which is heated over a water-bath 
for five to seven minutes. Take the dish containing the 
preparation off the water-bath and as soon as it becomes 
slightly opalescent, as the result of cooling, remove the 
cover-glass preparation and wash thoroughly in water. 
Then heat a few drops of the ethylamine silver solution 
upon the mordanted cover preparation until it just steams 
and the margin appears black. Next wash thoroughly in 
water and mount. 

This method is highly recommended by Stitt. 

The Staining of Capsules of Bacteria. — W. H. 
Smith's Method for Smears. — 1. Make a thin cover- 
glass preparation from fresh sputum, or pneumonic, pleural, 
or pericardial exudate. 

2. Pass through flame. 

3. Cover with a 10 per cent, aqueous solution of phos- 
phomolybdic acid (Merck) four to five seconds. 

4. Wash in water. 

If the micro-organism is Gram-staining, like the pneumo- 
coccus or streptococcus capsulatus, proceed as follows: 

5. Aniline oil methyl-violet, steaming one-quarter to one- 
half minute. 

6. Wash in water. 



METHODS OF STAINING BACTERIA. 245 

7. Treat with Gram's solution of iodin, steaming one- 
quarter to one-half minute. 

8. Decolorize with 95 per cent, alcohol. 

9. Wash in water. 

10. Stain with a 6 per cent, aqueous solution of eosin 
(Griibler's eosin, w.g.) one-half to one minute, warming gently. 

11. Wash in water. 

12. Wash in absolute alcohol. 

13. Clear in xylol and mount in xylol balsam. 

The capsule will be found to be distinct, clear cut, eosin- 
stained about the Gram-stained micro-organism. 

If the micro-organism is Gram-decolorizing, after step 4 
above proceed as follows : 




L A „ _ .. . . J 

Fig. 29. — Capsulated micrococci in a cover-glass preparation from an endo- 
carditic vegetation stained by W. H. Smith's method; X 2000 (W. H. Smith; 
photo, by L. S. Brown). 

5. Stain with a 6 pei cent, aqueous solution of eosin one- 
half to one minute, warming gently. 

6. Wash in water. 

7. Counterstain with Loffler's methylene-blue solution 
one-quarter to one-half minute, warming gently. 

8. Wash in absolute alcohol. 

9. Clear in xylol and mount in xylol balsam. The cap- 
sule will appear eosin-stained about the blue-stained micro- 
organism. 

Hiss's Method. — Dry the smear preparation in the 
air and fix by passing two or three times through the flame. 
Cover with a mixture of 5 c.c. of saturated alcoholic solu- 
tion of fuchsin or methyl-violet and 95 c.c. of distilled water 
and warm until steam appears. Wash off the mixture 
with 20 per cent, aqueous solution of sulphate of copper. 
Dry without washing in water. The capsule is blue and 
the body of the bacterium purple. 



246 PATHOLOGICAL TECHNIQUE. 

Richard Muir's Method. — I. The preparation is dried 
and stained in filtered carbol-fuchsin for half a minute with 
gentle heat. 

2. Wash slightly with methyl-alcohol and then well in 
water. 

3. Place in the following mordant for a few seconds: 

Saturated solution corrosive sublimate, 2 parts; 

Tannic acid solution, 20 per cent., 2 

Saturated solution of potash alum, 5 

4. Wash well in water. 

5. Treat with methyl-alcohol for about a minute. The 
preparation has a pale reddish appearance. 

6. Wash well in water. 

7. Counterstain with a watery solution of ordinary meth- 
ylene-blue for half a minute. 

8. Dehydrate in alcohol, clear in xylol, and mount in 
balsam. 

The bacteria are stained a deep crimson and the capsules 
a blue tint. The capsules of bacteria in cultures may some- 
times be demonstrated by this method. 

THE STAINING OF BACTERIA IN SECTIONS. 

Bacteria are demonstrated in sections of tissues almost 
entirely by means of the aniline dyes, of which three have 
thus far proved themselves to be particularly valuable, 
namely, methylene-blue, fuchsin, and methyl-violet. Of 
each of these dyes, one or more solutions have become 
famous because of their efficacy in staining and their keep- 
ing qualities. They will be referred to later. 

All bacteria yet known will stain when placed in appro- 
priate staining solutions. Some, however, are stained 
quickly, while others are stained with difficulty; some give 
up the stain readily to decolorizers, while others retain it 
tenaciously. In consequence of their reactions to certain 
dyes and to certain decolorizers, bacteria, from the point 
of view of staining, may be divided into two main groups: 

1. Bacteria which do not stain by Gram; 

2. Bacteria which stain by Gram. 



METHODS OF STAINING BACTERIA. 2Afi 

Included in the second group is a small group of acid-fast 
bacilli which can be stained in a special way by what is 
known as the tubercle-bacillus method. 

The organisms of the second group are much more easily 
demonstrated in tissues than those in the first group, 
because it is possible to stain them of one color and the 
nuclei of the cells of another color. In other words, it is 
possible to stain them so that they are differentiated from 
the issue in which they lie, and hence stand out prominently. 

The organisms of the first group have no differential stain ; 
they take the same color as the nuclei of the tissue. More- 
over, although they stain easily, most of them do not stain 
deeply, and readily part with the color they have taken up. 

It has been customary in the past to fix all tissues in 
which bacteria were to be demonstrated in alcohol. Of 
recent years formaldehyde has been much used for the same 
purpose. Zenker fixation can be recommended as being 
superior to either because of its perfect preservation not 
only of all bacteria but also of the tissues, so that by means 
of proper staining both the pathogenic organism and the 
lesion it produces can be perfectly and faithfully demon- 
strated. 

Sections which are to be stained for bacteria may be 
divided into two classes: paraffin sections and celloidin 
sections. 

Paraffin sections should, as a rule, be attached to the slide 
by means of Mayer's glycerin-albumin mixture. 

Celloidin embedding is to some extent a drawback to the 
stains for certain organisms, because the celloidin tends to 
hold the color, so that the bacteria are not so distinct as they 
otherwise would be. Still, it is so important to be able to 
stain bacteria in celloidin sections that particular care is de- 
voted in the following pages to methods which obviate most 
of the difficulties. 

It will usually be found advisable to attach celloidin sec- 
tions to the slide by means of ether-vapor. They will then 
keep perfectly flat in any staining solution, and may be 
heated without danger of wrinkling or contracting. The 
heat should never be applied directly under a section, but at 
one end of the slide. 



248 PATHOLOGICAL TECHNIQUE. 

Pathogenic Bacteria which do not Stain by Gram. 
(See page 236.) 

The staining solutions and methods employed for demon- 
strating this group of bacteria are applicable also to most 
of the organisms in the group which stain by Gram's method, 
and are, therefore, of great importance. 

Of the bacteria which do not stain by Gram or by the 
tubercle bacillus method, certain ones deserve special men- 
tion on account of their frequent occurrence or on account 
of the difficulty of demonstrating them in tissues, and 
certain variations in staining methods which have proved 
serviceable will be given. Loffler's methylene-blue solution 
has in the past been generally considered the most useful 
stain for this class of bacteria, but better results can be 
obtained by two other methods. The first is by means of 
the eosin-methylene-blue stain after Zenker fixation, a 
method which can be most highly recommended not only 
on account of the staining of the bacteria but also for the 
faithful demonstration of the histological changes present 
in the tissues. The second method is by means of Wol- 
bach's modification of Giemsa's stain which also gives most 
excellent results after Zenker fixation. Both of these stain- 
ing methods are given elsewhere. (See pages 74, 393.) 

Two other methods are added because they are some- 
times useful. 

Loffler's Methylene-blue Stain for Bacteria. — 1. Stain 
paraffin sections twenty minutes to twenty-four hours. 

2. Wash in weak acetic acid, 1 : 1000, for ten to twenty 
seconds. 

3. Absolute alcohol, two or three changes, to differentiate 
and dehydrate (as a rule, only a few seconds are required for 
this step). 

4. Xylol. 

5. Xylol balsam. 

For celloidin sections use 95 per cent, alcohol ; blot, and 
treat with xylol ; repeat until sections are clear ; mount in 
xylol balsam. 

This solution of methylene-blue is extremely useful, be- 
cause it will stain all bacteria except the tubercle-bacillus 



METHODS OF STAINING BACTERIA. 249 

group. Other solutions which may be used in the same 
way are — aniline-methyl-violet, Stirling's solution of methyl- 
violet, simple aqueous solutions of methyl-violet, and Ziehl's 
carbol-fuchsin. 

Methyl-green-pyronin Stain (Unna-Pappenheim) as modi- 
fied by Saathoff for bacteria: 

Methyl-green, 0.15; 

Pyronin, 0.50; 

96 per cent, alcohol, 5-00; 

Glycerin, 20.00 ; 

2 per cent, carbol-water ad 100.00. 

Stain sections two to four minutes, then wash in water, 
dehydrate quickly in absolute alcohol, clear in xylol, and 
mount in balsam. If acetone is used instead of absolute 
alcohol for dehydration there is less danger of decolorizing 
the cystoplasm of the cells. 

Pathogenic Bacteria which Stain by Gram. 
(See page 237.) 

These organisms, with the exception of the tubercle- 
bacillus group, are all readily stained by the general methods 
employed for staining under Group 1. For staining most 
of them in sections, however, the differential Gram-Weigert 
method will be found to give the most satisfactory results. 

The Gram Staining- Method. — Directions for staining 
paraffin sections: 1. Stain in aniline-methyl-violet five to 
twenty minutes. 

2. Wash in normal salt solution or water. 

3. Iodin solution (1:2: 300) one minute. 

4. Wash in water. 

5. Absolute alcohol, several changes, until no more color 
is given off and the section is apparently decolorized. 

6. Xylol. 

7. Xylol balsam. 

This method is not suited for celloidin sections, because 
the alcohol does not decolorize the celloidin sufficiently. In 
fact, it is better to reserve Gram's method for cover-slip 



250 PATHOLOGICAL TECHNIQUE. 

work alone, and to use instead of it, for sections of all kinds, 
Weigert's modification. This consists simply in the use of 
aniline oil instead of alcohol as a decolorizer. The method 
is easily acquired, is perfectly adapted not only to paraffin 
but also to celloidin sections, and the results are more 
perfect than after Gram. 

The Gram-Weigert Staining Method. — Preferably 
Zenker's fixation, paraffin sections: 

1. Stain sections lightly in alum-hematoxylin. 

2. Wash in running water. 

3. One per cent, aqueous solution of eosin soluble in 
water, one to five minutes. 

4. Wash in water. 

5. Aniline methyl-violet, one-half to one hour. 

6. Wash off with water. 

7. Lugol's solution, one to two minutes. 

8. Wash off with water. 

9. Blot with filter-paper and dehydrate and clear in 
several changes of aniline and xylol, equal parts. 

10. Wash off with xylol. 

11. Mount in xylol-colophonium. 

Verhoeff's Modified Gram Stain for the Leptothrix of 
Parinaud's Conjunctivitis. — He finds it superior to the 
ordinary methods for staining Gram-positive bacteria and 
leptothrix in sections. Zenker's fixation preferred. Either 
celloidin or paraffin sections may be used. 

1. Stain lightly in alum-hematoxylin and eosin, mount 
in Canada balsam. 

2. After five minutes or longer (ten years is not too long) 
remove cover-slip, by aid of heat if necessary, and wash off 
excess of balsam with xylol. If celloidin section, remove 
from slide. Chloroform, 95 per cent, alcohol, water. 

3. Stirling's gentian violet, twelve minutes. 

4. Water. 

5. Lugol's solution (1:2: 100), twenty seconds. Water. 

6. 95 per cent, alcohol, twenty seconds. 

7. Chloroform, fifteen seconds. 

8. Oil of origanum, fifteen seconds. 

9. 95 per cent, alcohol, thirty seconds. This removes 
excess of stain from celloidin. 



METHODS OF STAINING BACTERIA. 25 I 

10. Oil of origanum. If celloidin section, place on slide 
and blot. 

n. Wash off with xylol and blot. 

12. Mount in xylol-balsam. 

Great care is required in the differentiation in alcohol 
and chloroform. A variation of a few seconds here makes 
a great difference in the results. It is therefore well to 
carry a number of sections through Step 5 and then dif- 
ferentiate each separately, varying the time a few seconds 
from that stated. If the differentiation is perfect, the 
leptothrix filaments as well as the dots in them will be 
stained, otherwise only the dots may be stained, so that the 
organisms will appear as rows of dots. If the differentia- 
tion is carried too far, especially in the 95 per cent, alcohol 
(6), the organisms may be completely decolorized. In the 
case of Gram-positive bacteria the preliminary treatment 
with balsam may be omitted and the differentiation may 
be carried much further without danger of decolorizing the 
organisms. 

THE STAINING OF CAPSULES IN SECTIONS. 

William H. Smith's Method for Sections. — 1. Fixation 
in Zenker's fluid and paraffin sections. 

2. Cover with anilin-methyl-violet solution for a few 
seconds, warming by drawing the slide through the flame 
two or three times. 

3. Wash with Gram's I. K. I. solution. 

4. Wash with formalin (40 per cent, formaldehyde solu- 
tion). 

5. Decolorize with 95 per cent, alcohol. 

6. Wash quickly with Gram's I. K. I. solution. 

7. Cover with a special eosin mixture (see below), warm- 
ing in the flame for a few seconds. 

8. Wash, dehydrate with alcohol, clear with xylol, and 
mount in balsam. 

To obtain the best results the duration of the application 
of the various reagents must be varied with each prepara- 
tion, and in some instances, where very deep staining is 
desired, the stronger solution of I. K. I., Lugol's solution, 
may give better results. 



252 PATHOLOGICAL TECHNIQUE. 

The decolorization by alcohol may have to be supple- 
mented by washing with ether or with aniline-xylol, for the 
Gram staining may be so intense as to mask the red stain- 
ing capsules. This is particularly true in the case of Strep- 
tococcus viridans or streptococci in certain cases of endo- 
carditis in which a very narrow capsule may be demon- 
strable by this method. 




Fig. 30. — Streptococci with capsules in a section of lung; X 2000 (W. H. Smith 
and L. S. Brown). 

The special eosin mixture is made by shaking 1 part of 
aniline green in 200 parts of a 3 to 6 per cent, aqueous 
solution of yellowish water-soluble eosin and, after one or 
two hours' standing, filtering to remove the precipitate. 



PATHOGENIC BACTERIA AND FUNGI. 



The number of species of bacteria of pathogenic signif- 
icance which are commonly encountered in pathological pro- 
cesses in man is a small one. These comprise the staphylo- 
coccus pyogenes aureus, the streptococcus pyogenes, the 
pneumococcus, the bacillus coli communis, the typhoid ba- 
cillus, the bacillus diphtheriae, and the bacillus tuberculosis. 
It is with infections with these few species that the patholo- 
gist is most frequently concerned, and the determination of 
the presence of these alone comprises by far the greater part 
of the bacteriological work which he is called upon to do. 

In the following descriptions of the important pathogenic 
bacteria which are concerned in human pathology the main 
object will be to give those characteristics which will serve 
for their identification, rather than an exhaustive consideration 
of their various properties and modes of growth. 

Staphylococcus Pyogenes Aureus. — Morphology. — 
Rather small cocci, frequently arranged in masses or clumps. 

Stained by Gram's method. 

Blood-serum. — The colonies are golden yellow in color. 
They are rounded, shining, slightly elevated, and may 
attain a diameter of 2 mm. or more after remaining for 
thirty-six hours in the incubator. The color of the colo- 
nies varies from a pale yellow to a deep orange. Young 
colonies may be creamy white, becoming yellow later. 

Gelatin Stab-culture. — Growth along the line of stab, fol- 
lowed by liquefaction in funnel form, with yellow sediment 
and clouding of the liquefied medium (Fig. 32). 

Potato. — Yellow confluent colonies. 

Agar-agar Slant. — Rather broad shining streak with 
sharply defined margins, at first white in color, but later 
becoming yellow. 

Bouillon. — Densely clouded. A yellowish sediment is 
formed, and sometimes a thin pellicle is seen on the surface. 

253 



254 PATHOLOGICAL TECHNIQUE. 

Litmus-milk. — Turned pink and coagulated. 

Pathogenesis. — When inoculated into the circulation of a 
rabbit death follows in from eighteen hours to three days in 
the case of virulent cultures. Not all specimens of this 
organism are virulent. The lesions produced in the rabbit 
by inoculation in the ear-vein in typical cases are abscesses 
with infarctions in the kidneys, and miliary abscesses in the 












FlG. 31.— Staphylococcus pyogenes aureus from a culture; X 2000 (Wright and! 

Brown). 

myocardium, diaphragm, and voluntary muscles. In the 
kidneys lines of necrosis with purulent infiltration, mainly in 
the pyramids, are frequently observed. This organ is the 
one most constantly affected. The number and extent of 
the lesions vary in different animals and with different cul- 
tures. They are best developed in animals which survive 
about three days. In animals which succumb after eighteen 
hours no macroscopic change may be apparent. On micro- 
scopical examination of the kidneys, however, small areas of 
necrosis will usually be found, mainly in the pyramids, sur- 
rounding masses of cocci. In the kidneys of animals which 
survive longer all the grades of invasion of these necrotic 
areas by leucocytes, up to regular abscess-formation, may be 
traced. By cultures the organism will be found in large 



PATHOGENIC BACTERIA AND FUNGI. 



255 



numbers in the kidneys and urine of the rapidly fatal cases ; 
and in smaller numbers in the other organs and blood of the 
heart. 

Occurrence. — The staphylococcus pyogenes aureus is found 
most commonly in pus-formations of a circumscribed cha- 
racter and also in a large number of pathological conditions, 
of which only the more important will be mentioned here. 

These are as follows: Osteomyelitis, peritonitis, pleuritis, 




Fig. st. 



-Staphylococcus pyogenes aureus : stab-culture three days old in gela- 
tin (Fr'ankel and Pfeiffer). 



endocarditis, meningitis, broncho-pneumonia, and puerperal 
septicemia. It may also be found in the blood of the various 
internal organs at autopsies in cases in which a suppurative 
or other acute inflammatory process is present anywhere, 
with or without metastatic abscess-formation. The organism 
also occurs frequently in the dust of places inhabited by 
man, as well as on the surface of the skin and of the mu- 
cous membranes of the nose and mouth. 



256 



PA THOLOGICAL TECHNIQ UE. 



Diagnosis. — The staphylococcus pyogenes aureus cannot 
usually be identified with any certainty by the cover-glass 
examination alone. Cultures are necessary in order to dif- 
ferentiate from the other staphylococci and from the strep- 
tococcus. 

For practical purposes the identification of the pyogenic 
cocci may be made by the appearances of their colonies on 
blood-serum and by their morphology ; no secondary cult- 
ures are usually necessary. 

The following staphylococci may also be present in acute 
inflammatory processes, but they occur less frequently than 
does the staphylococcus pyogenes aureus. 

Staphylococcus Pyogenes Albus and Staphylococcus 
Pyogenes Citreus. — These organisms differ from the staphylo- 
coccus pyogenes aureus mainly in the color of their colonies. 
As a rule, they are much less pathogenic for rabbits than that 
organism. 

Staphylococcus Epidermidis Albus (Welch). — " Is prob- 
ably only a variety of the staphylococcus pyogenes albus. Usu- 




FiG. 33. — Streptococcus pyogenes ; cover-glass preparation of the pus of an 
abscess ; X 1000 (Frankel and Pfeiffer). 



ally grows somewhat more slowly ; liquefies gelatin and coagulates 
milk less rapidly. Is of little virulence under ordinary conditions. 
Is a regular inhabitant of the epidermis, lying deeper than can be 
reached by disinfection of the surface of the skin" (Welch). 



PATHOGENIC BACTERIA AND FUNGI. 



257 



Staphylococcus Cereus Albus. — Very similar to the staph- 
ylococcus pyogenes albus, but does not liquefy gelatin. May 
occur in abscesses. 

Staphylococcus Cereus Plavus. — This organism is similar 
to the preceding, except that it forms a lemon-yellow pigment. 

Streptococcus Pyogenes and Allied Streptococci. 

— Different strains of streptococci show considerable varia- 
tion in their biological properties. It is not certain whether 
those concerned in pathological processes are of various 
species or only modifications by environment of one species. 
Morphology. — Rather small cocci arranged in chains, many 
of the cocci being divided into two hemispheres by a line 
of division running at right angles to the axis of the chain. 
In some strains the cocci are conical or oval in outline. The 
chains may be made up of many cocci and be quite long. 




Fig. 34. — Streptococcus pyogenes from a culture in bouillon; x 2000 (Wright 

and Brown). 

Cover-glass preparations from the colonies often fail to 
show the characteristic chain arrangement, owing to the 
chains being broken up by the manipulation. The chain- 
formation is best demonstrated in cover-glass preparations 
from the " water of condensation " at the bottom of the 
blood-serum tube. This is essentially a bouillon culture, 
and it is in such fluid media that the chain-formation is best- 
developed. In preparing the cover-glass from this as little 
manipulation of the fluid as possible should be used, in 
order to avoid destroying the chain arrangement. 

17 



258 PATHOLOGICAL TECHNIQUE. 

Practically, the only organism with which the strepto- 
coccus may be confounded is the pneumococcus, which also 
grows in minute colonies and sometimes in chains. The 
streptococcus may be distinguished from the pneumococcus, 
however, by the morphology of the individual organisms, 
the streptococci appearing as pairs of hemispheres, and the 
pneumococci as pairs of oval, conical, or lancet-shaped or- 
ganisms, the broader ends of which are in apposition. 
It also differs from the pneumococcus in that it does not 
dissolve in bile and does not ferment inulin. 

Stains by Gram's method. 

Blood-serum. — Minute grayish- white colonies, often look- 
ing like small grains of sand scattered over the surface of 
the medium. Sometimes the colonies are shining, trans- 
lucent, colorless, resembling minute dewdrops. 

Bouillon. — The character of the growth in bouillon is sub- 
ject to considerable variation, and certain doubtful varieties 
of the streptococcus are distinguished mainly by the bouil- 
lon culture. 

" We thus distinguish short-chained streptococci (' strepto- 
coccus brevis '), long-chained streptococci (' streptococcus 
longus '), streptococci which render bouillon cloudy and 
those which do not, streptococci which form flocculent or 
scaly or sandy or viscous sediments. 

" The name ' streptococcus conglomeratus ' is given to a 
streptococcus which grows, without clouding the bouillon, in 
the form of dense, separate particles, scales, or thin mem- 
branes at the bottom or sides of the tube, and on shaking 
the sediment it breaks up into little specks, without produ- 
cing uniform diffuse cloudiness. 

" On microscopical examination the chains in the latter 
case are long and interwoven in conglomerate masses. 
Streptococcus chains may be straight or wavy or twisted. 
These various distinctions are only of relative value. One 
form may change into another. Virulent streptococci may 
be found among all the groups mentioned ; the streptococci 
of erysipelas and most of the streptococci from abscesses 
and septicemia grow in long chains in bouillon " (Welch). 

Ascites Dextrose Bouillon. — This consists of 1 part of 



PATHOGENIC BACTERIA AND FUNGI. 



259 



sterile ascitic fluid mixed under aseptic precautions with 3 
or 4 parts of sterile I per cent, dextrose bouillon. In this 
medium the streptococci grow more abundantly than in 
ordinary bouillon. 

Agar-agar Slant. — Minute grayish translucent colonies 

( Fi g- 35)- 

Agar-agar Stab. — Small spherical grayish colonies along 

the needle-track. 

Gelatin. — Growth similar to that on agar-agar. 

Litmus-milk. — Some varieties turn 
the medium pink and cause coagu- 
lation. 

Fermentation of Carbohydrates. — 
Great differences exist among various 
strains in the ability to ferment certain 
carbohydrates, such as lactose, man- 
nite, salicin, dextrose, and saccha- 
rose. One or more of these or other 
carbohydrates may or may not be 
fermented by a given strain. 

For testing fermentation, Hiss's 
serum-water medium may be used. 
This consists of a mixture of 1 part of 
beef serum and 2 or 3 parts of distilled 
water, to which 1 per cent, of a 5 per 
cent, solution of purified litmus is 
added. This mixture is heated to 
ioo° C. for a few minutes, and then 
the carbohydrate desired is added to 
it in the proportion of 1 per cent., after 
which it is distributed in fermenta- 
tion tubes and sterilized in the usual 
manner. Instead of the fermentation 
tubes, Durham's tubes may be used, 
culture-tubes, in each of which is placed, in inverted position, 
a small test-tube about 7 cm. long and 8 mm. in diameter. 
The outer tubes are filled with the medium to a height of 
about 5 cm. During sterilization the air in the inner tubes 
is driven out, and, upon cooling, the medium takes its place. 




Fig. 35. — Streptococcus 
pyogenes : culture upon 
agar-agar two days old 
(Frankel and Pfeiffer). 

These are ordinary 



26o PATHOLOGICAL TECHNIQUE. 

Fermentation is indicated by the appearance of gas in the 
inner tube. 

Hemolysis. — Some strains hemolyze human red blood-cor- 
puscles and some do not. Hemolyzing power is tested by 
growing colonies upon the surface of blood-agar plates. 
Hemolysis is shown by the appearance of a clear zone about 
the colony. Some strains cause a greenish coloration of the 
surrounding medium, and are called by some writers Strep- 
tococcus viridans. The blood-agar plates may be made as 
described for hydrocele fluid-agar plates on page 273. Sterile 
defibrinated human blood is used instead of hydrocele fluid. 

Pathogenesis. — The results of the inoculation of animals 
are not constant, great variation in the virulence of dif- 
ferent cultures being observed. Sometimes mice inoculated 
at the root of the tail or in the peritoneal cavity will die in 
about twenty-four hours with enlargement of the spleen and 
large numbers of the organism in the internal organs. 

Some strains produce arthritis in rabbits by intravenous 
inoculation. E. C. Rosenow has shown also that the Strep- 
tococcus viridans may produce vegetative endocarditis in these 
animals. He injected intravenously large quantities of the 
micro-organism obtained by centrifugalization of cultures in 
ascites-dextrose bouillon, and used young animals. With 
strains from the joints of acute articular rheumatism he pro- 
duced arthritis, endocarditis, pericarditis, myocarditis, myo- 
sitis, and other lesions. 

Occurrence. — The streptococcus occurs frequently in the 
spreading phlegmonous inflammations as well as in suppu- 
rative processes generally, and is the most common cause 
of septicemia. It is almost always present in inflammatory 
conditions of the mucous membrane of the pharynx, and is 
often encountered in bronchopneumonia. In erysipelas it 
is almost invariably the infecting organism, and it is the 
most frequent cause of puerperal septicemia. In the joints 
of acute rheumatism the streptococcus has been demon- 
strated. In the majority of fatal cases of diphtheria and 
in some cases of typhoid fever, scarlet fever, tuberculosis, 
and other acute inflammatory diseases it will be found in 



PATHOGENIC BACTERIA AND FUNGI. 26 1 




Fig. 36. — Streptococcus viridans from a culture; X 1503 (W. H. Smith and L. S. 

Brown). 







Fig. 37. — Streptococcus viridans in a section from a cardiac vegetation; X 1000 
(W. H. Smith and L. S. Brown). 



262 PATHOLOGICAL TECHNLQUE. 

the blood of the various internal organs after death. It also 
occurs in a certain proportion of cases of peritonitis, pleu- 
ritis, meningitis, endocarditis, and otitis media. In endocar- 
ditis, masses of the micro-organisms may form a large part 
of the substance of the cardiac vegetations. Gaining entrance 
to the tissues through an insignificant wound or abrasion of 
the skin, it may produce a rapidly fatal septicemia in a sus- 
ceptible individual, in whose internal organs at autopsy large 
numbers of the organism will be found. This general inva- 
sion of the circulation may also be observed in cases of 
chronic or wasting disease, the infection occurring during 
the last days or_ hours of life (terminal infection). 




Fig. 38. — Streptococcus viridans showing capsules in a section of a cardiac vegeta- 
tion; X 1500 (W. H. Smith; photo by L. S. Brown). 

Of other conditions in which it may occur, hepatic abscess, 
appendicitis, osteomyelitis, and synovitis may be mentioned. 
Although the streptococcus is distinctly one of the pus-pro- 
ducing bacteria, yet the inflammations of the soft parts of 
the extremities which are produced by it are generally cha- 
racterized more by necrosis and serous or hemorrhagic exu- 
dation and infiltration than by the breaking down of tissue 
and frank pus-production. In this the organism is in marked 
contrast to the staphylococcus pyogenes aureus, which 
practically always produces dissolution of tissue and pus. 
Moreover, the streptococcus inflammations are more com- 
monly accompanied by lymphangitis than are those due to 
the staphylococcus pyogenes aureus. 



PATHOGENIC BACTERIA AND FUNGI. 263 

In a few instances we have met with a streptococcus whose 
colonies assume a well-marked yellow color on blood-serum, but 
which in other respects are like the long-chained forms above de- 
scribed. 

Diagnosis. — The streptococcus pyogenes may often be 
identified by the cover-glass examination alone through its 
characteristic chain-formation, but this may not be apparent 
and the result of cultures must then be awaited. 

. Cultures from the blood during life should be made in 
ascites- dextrose bouillon (see page 258). 

For cultures from joints in acute rheumatism, E. C. Rose- 
now recommends aspirating the joint with a sterile syringe, 
and mixing 1 c.c. or less of the joint fluid with fluid ascites- 
dextrose agar, cooled to 40 C. After the tubes have been 
inoculated and their contents mixed, they are to be placed 
in cold water to solidify the agar and then in the incubator. 
In this way the streptococci are given a chance to grow with- 
out oxygen in the depths of the medium, a condition which 
favors their growth. 

The ascites-dextrose agar tubes are prepared as follows : 
2 per cent, nutrient agar containing 0.2 to 1 per cent, of dex- 
trose, of a reaction 0.4 to 0.6 per cent, acid to phenolphthalein, 
is boiled in tubes containing 7 to 8 c.c. each for a few minutes 
to expel absorbed oxygen, cooled to 50 C, and then to 
each tube is added 2 or 3 c.c. of sterile ascitic fluid which has 
been previously heated to 60 ° C. for twenty-four hours. The 
tubes are then cooled to 40 C. and inoculated as above 
described. 

In erysipelas the streptococcus is most readily found in 
the extreme margin of the affected area where the process is 
newest. The skin should be cleansed with soap and water, 
and with alcohol. Then with a sterile knife-point or a large 
needle a small wound should be made, and some of the 
blood and exudate pressed out from the tissue beneath. 
From this cultures and cover-glasses may be prepared. 

Pneumococcus. — Synonyms : Diplococcus pneumoniae ; 
Micrococcus lanceolatus ; Micrococcus of sputum-septi- 
cemia; Micrococcus pneumoniae crouposae. 



264 



PA THOL O GICAL TE CHNIQ UE. 



Morphology. — Pairs of rather small oval, conical, or lan- 
cet-shaped organisms, the broader ends being in apposition. 
The organism varies somewhat in size, and one of the "pair ,: 




Fig. 39. — Pneumococci with capsules in a cover-glass preparation from peri- 
cardial exudate stained by W. H. Smith's method ; X 1500 (W. H. Smith ; photo. 
by L. S. Brown). 

may be smaller than the other (Fig. 39). In some cases 
atypical or involution forms are seen, especially if the culture 
be more than twenty-four hours old. No capsules are ordi- 




Fig. 40. — Pneumococci from a culture ; x 2000 (Wright and Brown). 

narily observed in cultures with ordinary methods of staining 
In the " water of condensation " of the blood-serum tube, 
chains may be formed resembling those of the streptococcus, 
but differing from the chains of that organism by the oval or 
lancet form of the elements of which they are composed. 



PATHOGENIC BACTERIA AND FUNGI. 



265 



In pus, blood, or in other material the organism is invested 
with a hyaline zone, called the capsule (see Figs. 40, 41). 
This is composed of a mucin-like substance. It may be 
seen usually in cover-glass preparations stained by the or- 
dinary methods, especially if the preparations be examined 
in water-mounting. 

Stained by Gram's method. Not motile. 








Fig. 41. — Diplococcus pneumoniae ; cover-glass preparation from the heart's 
blood of a rabbit; x iooo (Frankel and Pfeiffer). 

Blood-serum. — Minute colorless, transparent colonies, re- 
sembling very small drops of dew (Fig. 42). 

Glycerin Agar-agar. — Feeble growth of very minute gray- 
ish colonies. 

Bouillon. — Clouded faintly. 

Litmus Milk. — Sometimes turned pink and coagulated. 
Growth on other culture-media is very feeble. The organ- 
ism dies out rapidly in cultures. To keep it viable it should 
be transplanted every forty-eight hours. 



266 PATHOLOGICAL TECHNIQUE. 

Pathogenesis. — The pneumococcus is very pathogenic for 
mice and rabbits, less so for guinea-pigs. 

Subcutaneous inoculation with virulent cultures causes the 
death of mice in from twenty-four to thirty-six hours, and 
of rabbits in from thirty-six to forty-eight hours, with septi- 
cemia. 

This infection is the " sputum-septicemia " of Sternberg. 
At the autopsy there will be found in the blood everywhere 
the characteristic encapsulated lancet-shaped organisms, usu- 
ally in pairs (Fig. 41). Great variation in the virulence of 
the organism is observed. In some cases no effect will be 
produced by the inoculation ; in others a more or less exten- 
sive fibrino-purulent exudation will be produced about the 
point of inoculation, and the animal will survive for a con- 
siderable length of time or recover. Inoculation into the 
ear-vein or peritoneal cavity of a rabbit will sometimes cause 
a rapidly fatal septicemia, when subcutaneous inoculation with 
the same culture will only cause a local reaction. The 
virulence of the pneumococcus is quickly lessened by culti- 
vation. 

Occurrence. — The pneumococcus may be demonstrated in 
the pulmonary exudate of practically all cases of genuine 
lobar or croupous pneumonia. At autopsies on cases of this 
disease it may be found in large numbers in the consolidated 
lung, and sometimes in smaller numbers in the blood of 
other internal organs. Cultures from the lung may some- 
times show the presence of other bacteria in addition to the 
pneumococcus, but these are to be regarded as either second- 
ary infections or contaminations from the smaller bronchi. 

The pneumococci in the pneumonic exudate die in large 
numbers after a time, and in cases near resolution numerous 
capsules may be found in cover-glass preparations from the 
lung in which it is impossible to demonstrate the organism 
by staining methods. 

The pneumococcus is also frequently found in broncho- 
pneumonia, acute peri- and endo-carditis, acute pleuritis and 
empyema, acute purulent meningitis, and in otitis media. In 
cases of pneumonia and bronchitis it may be present in the 






PATHOGENIC BACTERIA AND FUNGI. 267 

sputum in large numbers. It has been observed in cases of 
peritonitis, of synovitis, of osteomyelitis, and of abscess-forma- 
tion in various situations. 

At autopsies on individuals dead of these conditions it 
ma)/ be frequently found, by means of cultures and animal 
inoculations, generally distributed throughout the internal 
organs in variable numbers. It is also often present in the 
mouth and in the saliva of healthy individuals. 



/ 



WJ 



M 
* 

f 

* 
» 


i / 


» 1 


1 1 




/ 



FlG. 42. — Streptococcus capsulatus in cover-glass preparation from sputum stained 
by W. H. Smith's method (W. H. Smith ; photo, by L. S. Brown). 

Diagnosis. — If the pneumococcus be present in very small 
numbers in pathological material, the quickest and most 
certain method of demonstrating its presence is the inocula- 
tion of a mouse with some of the material (see page 231). 
This is also the best way to prove the identity of the organ- 
ism. In severe infections it may be demonstrated during 
life in the blood. 



268 PATHOLOGICAL TECHNIQUE. 

Culture-media should be 0.3 to 0.5 acid to phenolphtha- 
lein, should be made from meat infusion, not from beef 
extract, and should not be sterilized in the autoclave, but 
in the Arnold apparatus for twenty minutes on three succes- 
sive days. The addition of sterile defibrinated rabbit blood 
to bouillon and agar in the proportion of a few drops to 4 
or 5 c.c. favors the growth of the pneumococcus. 

The pneumococcus can usually be identified in exudates, 
blood, tissues, or sputum by examination of cover-glass 
preparations alone, by reason of its peculiar morphology and 
its possession of a capsule. The capsule can be seen in most 
instances in cover-glass preparations, stained in the usual 
manner, if they be examined in water-mount. The capsules 
appear as a hyaline material usually with definite outlines 
surrounding the paired organisms. It may be distinguished 
from the streptococcus by two characteristics, namely, that 
it is dissolved by bile and that it ferments inulin. 

The Determination of Types of Pneumococcus. — Re- 
searches carried on in the Rockefeller Institute for Med- 
ical Research have shown that several types of pneumococ- 
cus may be differentiated as the infectious agent in lobar 
pneumonia by agglutinative reactions with immune sera. 
The technique may be described as follows: 1 

A small coherent portion of sputum a few millimeters in 
diameter, which is known to be rich in pneumococci, after 
washing in sterile normal salt solution, is ground up in a 
sterile mortar with about 1 c.c. of sterile bouillon salt 
solution and about 1 ex. of the mixture is injected into the 
peritoneal cavity of a white mouse. 

When the mouse appears sick, which may occur after 
five to twenty-four hours, the belly is punctured with a 
capillary pipette and a smear preparation of a small drop 
of the peritoneal fluid examined. This procedure is re- 
peated later if the fluid be not rich in pneumococci, as it 
may be necessary to wait until the mouse dies. When the 
fluid has been found to contain sufficiently numerous pneu- 
mococci, the animal, if not already dead, is killed, the 

Abstracted from "Acute Lobar Pneumonia, Prevention and Serum 
Treatment," by Oswald T. Avery, M. D., H. T. Chickering, M. D., Rufus 
Cole, M. D., and A. R. Dochez, M. D. Monographs of the Rockefeller In- 
stitute for Medical Research, No. 7, October 16, 191 7. 



PATHOGENIC BACTERIA AND FUNGI 269 

peritoneal cavity opened and washed out, under sterile pre- 
cautions, with 4 or 5 c.c. of salt solution with the aid of a 
pipette provided with a rubber bulb. The washings are 
immediately transferred to a sterile tube and centrifugated 
at low speed for a short time to throw down cells and fibrin ; 
then the supernatant fluid is drawn off into another tube 
and centrifugated at high speed until the organisms are 
deposited as a sediment. From this sediment, after re- 
moval of the supernatant fluid, a suspension in salt solution 
is made of about the same concentration as that of a bouillon 
culture of pneumococcus. 

This suspension is tested for the type of pneumococcus 
which it may contain as follows: 

A row of five small tubes is set up. In each of the first 
four tubes is placed 0.5 c.c. of the suspension, but in the 
fifth tube only 0.4 c.c. Then there is run into tube 
No. I 0.5 c.c. of immune serum I, diluted 1 : 20; into 
tube No. 2 immune serum II, undiluted; into tube No. 3 
immune serum II, diluted 1 : 20; into tube No. 4, immune 
serum III, diluted 1:5; and into tube No. 5, 0.1 c.c. of 
sterile ox bile. The tubes are incubated at 37 C. and read 
after an hour. 

The identification of Types I, II, or III of the pneu- 
mococcus is made by the appearance of clumping or ag- 
glutination of the organisms in the tube containing the 
corresponding immune serum. If there is no agglutination 
in any of the tubes and the bacteria in the bile tube are 
found upon microscopical examination to be dissolved, then 
the organism is identified as Type IV pneumococcus. As 
all strains of pneumococcus are dissolved by bile and all 
strains of streptococci are not, the fifth tube serves to pre- 
vent the mistake of classifying a streptococcus as Type IV 
pneumococcus. The bile is prepared by autoclaving, filter- 
ing off the precipitate, and again autoclaving. 

Types I and II occur in about equal proportion in more 
than 60 per cent, of all the cases of pneumonia investigated. 

Type III is apparently identical with some of the strains 
of ''Streptococcus Capsulatus," the characters of which we 
describe elsewhere (see page 270), and occurs only in a 
small percentage of cases. 



270 



PATHOLOGICAL TECHNIQUE. 



Type IV occurs in about 20 per cent. 

For control, cultures on blood-agar plate and in bouillon 
should be made from the heart blood of the mouse, and with 
a pure bouillon culture thus obtained confirmation of the 
type should be carried out as in the case of the bacterial 
suspension described above. 

If a positive culture from the blood of the patient be 




FIG. 43- 



Fig. 44- 



Fig. 45- 



Fig. 43. — Pneumococcus ; blood-serum culture. 
Fig. 44. — Streptococcus capsulatus ; blood-serum culture. 
Fig. 45- — Streptococcus capsulatus; glucose-agar stab culture (Oscar Rich- 
ardson ; photos, by L. S. Brown). 



available, 10 c.c. of the fluid should be centrifugated and a 
suspension of the organism prepared and tested as described 
above. Likewise the determination of type may be made 
for pneumococci obtained by culture from spinal fluids and 
other material. 

Streptococcus Capsulatus. — This seems to be the best 
name to apply to a capsule-bearing Gram-positive bac- 
terium resembling both the pneumococcus and the strepto- 



PATHOGENIC BACTERIA AND FUNGI. 2*}\ 

coccus pyogenes in morphology, but differing definitely from 
them in cultural and other peculiarities. It has been found 
chiefly in lobar pneumonia, but occurs in other inflammatory 
processes and probably has been sometimes mistaken for 
the pneumococcus or the streptococcus pyogenes. 

Oscar Richardson points out the following chief character- 
istics by which it may be distinguished from the pneumo- 
coccus and the streptococcus pyogenes: 

1. The capsules persist in cultures (see Fig. 46). 

2. On the surface of coagulated blood-serum its colonies 
are flat, colorless, viscid, mucus-like, of irregular outline, 




Fig. 46. — Streptococcus capsulatus, from a blood culture in a case of pneu- 
monia; stained by W. H. Smith's method; X about 700 (W. H. Smith; photo 
by L. S. Brown). 

and may attain a diameter of 2 or 3 mm. They may become 
confluent and form large patches of mucus-like material 
(see Fig. 44). 

3. In glucose-agar stab, adjusted to a reaction of 0.5 acid 
to phenolphthalein,. there is growth all along the line of 
inoculation, from which, in places, fusiform or hemispherical 
masses of growth extend into the surrounding medium in a 
vertical plane, apparently occupying clefts in the medium 
(Fig. 45). It is very important for the development of 
these characteristic appearances that the glucose-agar be 



272 



PA THOLOGICA L TECHNIQUE. 



known to have at the time of inoculation a reaction very 
close to that above indicated. 

Gonococcus. — Morphology. — Cocci of medium size, com- 
posed usually of two hemispheres separated by a narrow 
unstained interval. Sometimes two of these pairs of hemi- 
spheres are joined together in the manner of "tetrads," or 
groups of four, showing evidence that division occurs in two 
directions at right angles to each other (Fig. 47). Decolor- 
ized by Gram's method. 

The gonococcus will not grow satisfactorily upon any of 
the culture-media ordinarily employed, but requires special 
media for its cultivation. 



* 








• 
• 


* % 




* 



Fig. 47. — Gonococci from a culture, showing formation of tetrads and variability 
in the size of the cocci; X 2000 (Wright and Brown). 

The colonies on suitable culture-media appear after eigh- 
teen to twenty-four hours as minute, grayish, translucent 
points. Later they may attain a diameter of 2 mm. Under 
low magnifying power a well-developed colony is seen to 
consist of a generally circular expansion, with thin, trans- 
lucent, sharply defined margins, becoming brownish, granu- 
lar, and denser toward the center, which is made up of coarse 
brownish clumps closely packed together (Fig. 48). 



PATHOGENIC BACTERIA AND FUNGI. 



273 



Special Culture-media. — The essential constituent of 
culture-media upon which the gonococcus will grow seems 
to be the blood-serum or similar albuminous fluid from the 
animal body. 

Probably the most convenient culture-medium for the 
cultivation of the gonococcus is hydrocele-fluid agar. This 
medium consists of sterile hydrocele fluid mixed with fluid 
agar-agar at a temperature of 40 C, in the proportion of 
I part of hydrocele fluid to 2 or 3 parts of agar-agar. The 
hydrocele fluid is to be obtained under the strictest precau- 
tions to avoid contamination with bacteria, thoroughly ster- 
ilized vessels, etc., being used. 

Ordinary tubes of plain agar-agar (2 per cent.) which have 
been previously sterilized in the usual manner are melted and 




Fig. 48. — Gonococcus colony (low magnifying power; photo by L. S. Brown). 

brought to a temperature of 40 C. in a water-bath. To the 
fluid agar-agar in each tube the sterile hydrocele fluid is then 
added in the proportion of one-third to one-half the volume 
of the agar-agar, care being taken to avoid contamination. 
For the transfer of the serum to the agar-agar tubes a steril- 
ized pipette may be used. The tubes may then be infected 
and their contents poured into sterilized Petri dishes, as in 
the plate method previously described (see page 218), or the 
tubes may be placed on their sides in a slightly inclined 

18 



274 PATHOLOGICAL TECHNIQUE. 

position and the agar-agar allowed to solidify, thus form- 
ing "slants" which may be kept on hand ready for use. In 
order to test for the presence of contaminating bacteria in 
these slants, it is well to place them in the incubator for 
twenty-four hours after they have become solid, so that any 
bacteria which may be present in them will form colonies 
and manifest themselves. Some pathological fluids which 
are rich in albumin, such as the serous exudate of pleuritis, 
may be used in the place of the hydrocele fluid as above 
described. 

Occurrence. — The presence of the gonococcus may be 
demonstrated in the pus of acute gonorrhea and gonorrheal 
ophthalmia. It occurs also in a certain proportion of cases 
of purulent salpingitis. It has been found in peritonitis, en- 
docarditis, pericarditis, myocarditis, pleuritis, and arthritis, as 
well as in peri-urethral abscess, in abscess of the glands of 
Bartholini, and in other acute inflammatory processes. In a 
few cases of endocarditis it has been demonstrated in the 
blood during life. 

Diagnosis. — For practical purposes the gonococcus may 
be sufficiently identified in pus by cover-glass examination 
of the same. Cocci in the form of paired hemispheres 
chiefly situated within the pus-cells and decolorizing by 
Gram's method of staining may be regarded as gonococci 
with a fair degree of certainty. 1 The fact that they decol- 
orize by Gram's method serves to distinguish them from 
the pyogenic staphylococci and streptococci, for these may 
also be present inside leucocytes, and may in some instances 
look like gonococci. The identification by this means is not 
beyond question. To make it more certain the isolation 
and study of the suspected cocci in cultures are necessary. 
In cultures, not only should the organism show the pecu- 
liarities of morphology, of staining, and of colony growth 
above described, but it should be rigidly determined that it 
does not grow on ordinary agar-agar. 

1 There is no trustworthy evidence that any other Gram-decolorizing micro- 
coccus than the gonococcus ever occurs in gonorrheal pus. F. T. Lord, work- 
ing in the laboratory of the Massachusetts General Hospital, examined by cul- 
tures the pus from 22 cases of gonorrhea, and in none could he find any 
Gram-decolorizing micrococcus other than the gonococcus. 



PATHOGENIC BACTERIA AND FUNGI. 



275 



In making transplants to plain agar avoid carrying over 
any of the albuminous material of the special culture- 
medium, for this material may permit some growth of the 
gonococcus on the plain agar. 

In applying the test of decolorization by Gram's method, 
colonies not more than forty-eight hours old should be used, 
because Gram-staining cocci in older cultures may be more 
or less decolorized by this method. In proof of the neces- 
sity of cultures for confirming the identity of the gonococcus 
in certain instances we may state that we have met with a 
Gram-decolorizing coccus in an arthritis of the knee, clini- 




FiG. 49. — Gonococci inside a leucocyte. Cover-glass preparation from gonor- 
rheal pus ; X 2000 (Wright and Brown). 

cally of gonorrheal origin, which, in cover-glass preparations 
from the exudate, was regarded as the gonococcus, but which 
was found not to be that organism by the study of it in 
cultures. 

If it is desired to obtain cultures of the gonococcus from 
the pus of gonorrheal urethritis, the case should not be more 
than of a few days' duration, because cases of longer dura- 
tion will usually show the presence of other bacteria whose 
colonies overgrow the feebly growing colonies of the gono- 
coccus. An organism which may be mistaken for the gono- 
coccus is a coccus growing in large milk-white colonies on 
all media, but staining by Gram's method. This coccus is 



276 



PA THOL O GICA L TE CHNIQ UE. 



frequently found in gonorrheal pus after the discharge has 
lasted several days. Other cocci also occur. 

The pus for culture purposes may be collected on a 
"swab," and the special culture-medium directly infected 
with this. The gonococcus retains its vitality in the pus 
on the swab for a number of hours, but care should be 
taken to avoid drying. 




Fig. 50. — Micrococcus catarrhalis in smear from sputum (F. T. Lord; photo by 

L. S. Brown). 

In a certain proportion of cases of purulent inflammation 
of the oviducts gonococci may be found and cultivated, as 
above indicated. The majority of cases, however, will have 
sterile pus, while in a small percentage of cases the ordinary 
pyogenic cocci will be present. 

Cultures from the blood in cases of gonorrheal endocar- 
ditis should be made in hydrocele bouillon. This consists 
of 1 part of sterile hydrocele fluid and 3 parts of sterile 
bouillon mixed under aseptic precautions. 

Method of Staining" for Gonococci. — Prepare a cover- 
glass with the pus, spreading it thinly with the platinum 



PATHOGENIC BACTERIA AND FUNGI 



277 



wire. The practice of spreading a small drop of pus between 
two cover-glasses and drawing them apart is objectionable. 
After ''fixing," stain the preparation by the following method: 

1. Stain with aniline-methyl-violet solution for thirty 
seconds, without heating. 

2. Wash in water. 

3. Coverthe preparation with Gram's 
iodin solution for thirty seconds. 

4. Wash in water. 

5. Wash with alcohol (95 per cent.) 
until the color ceases to come out of the 
preparation. 

6. Wash in water. 

7. Stain in saturated aqueous solution 
of Bismarck brown for thirty seconds. 

8. Wash in water and mount. 
This method is nothing but Gram's 

method and after staining with Bis- 
marck brown. With it the gonococci 
are stained brown, and other pyogenic 
cocci blue-black. 

W. F. Whitney has suggested the 
use of a 1 per cent, aqueous solution 
of pyronin in place of the solution of 
Bismarck brown in step 7. The gono- 
cocci are stained red by the pyronin. 

Pappenheim's Method. — 1. Stain 
smear preparation for three to five 
minutes in the following mixture: 
Methyl green, 
Pyronin, 
5 per cent, carbolic acid water, 

2. Wash off in water; dry; mount in xylol balsam. 

Nuclei green; cocci bright red. 

For demonstrating the gonococcus in sections of tissues 
the general stains used for Gram-negative bacteria give 
good results. After Zenker's fixation Mallory's eosin and 
methylene-blue method is recommended. 

Micrococcus Catarrhalis. — This micrococcus may be 
found in the sputum in inflammatory conditions of the 




Fig. 51. — Micrococcus 
catarrhalis colonies on 
agar (F. T. Lord ; photo 
by L. S. Brown). 

2 grams; 
2 " 
IOO c.c. 



278 



PA THOL O GICAL TE CHNIQ UE. 



respiratory tract and cannot be distinguished in its mor- 
phology and staining reactions from the gonococcus or from 
the Diplococcus intracellularis meningitidis (Fig. 53). The 
appearances of its colonies on ordinary culture-media are, 
however, characteristic. They are large, white, of irregular 
outline, and have elevated central portions. They are friable, 
not viscid, and grow readily at room- temperature (Fig. 51). 

Micrococcus TetragentlS. — The colonies are small, 
white, and elevated. Growth is slow. 

Morphology. — Micrococci arranged in fours, or " tetrads," 
held together by a gelatinous substance (Fig. 52). 

Stained by Gram's method. Not motile. 

Gelatin Stab. — Feeble growth in the form of minute spheri- 
cal masses along the line of stab with a small white, slightly 
elevated point at the surface of the medium. The gelatin 
is not liquefied. 



?*F(r '.•• 




.*.«« 



Fig. 52. — Micrococcus tetragenus in pus from a white mouse; X & l 5 (Heim). 

Agar-agar Slant. — Moist, glistening, grayish-white trans- 
lucent streak with wavy margins. 

Potato. — Growth is in the form of a thick, irregular, slimy- 
looking patch. The growth on agar-agar and on potato 
may be drawn into thin threads by the platinum wire. 

Pathogenesis. — Subcutaneous inoculation of mice and 
guinea-pigs may lead to a fatal septicemia or only a local 
pus-formation. Intravenous or intraperitoneal inoculation 
of rabbits may also produce septicemia and death. 



PATHOGENIC BACTERIA AND FUNGI. 279 

At autopsy the micrococci, arranged in tetrads, are found 
in the blood generally, but most numerously in the spleen. 
They can readily be demonstrated by cover-glass prepara- 
tions. 

Occurrence. — " Found not infrequently in phthisical cavi- 
ties and sputum, occasionally in association with pyogenic 
cocci in abscesses connected with carious teeth and about 
the neck and jaws and middle ear, rarely in abscesses else- 
where. It has been considered to be non-pathogenic for 
man, but it has been found in pure culture in closed ab- 
scesses in man, and Viquerst has proved experimentally 
that it is capable of causing suppuration in human beings " 
(Welch). 

Diplococcus Intracellulars Meningitidis. — Mor- 
phology, — Diplococci, occurring as paired hemispheres, 
separated by a well-marked unstained interval and showing 
considerable variation in size in cultures (Fig. 53). The 




larger forms are regarded as involution or degenerate forms. 
The organism shows a tendency to grouping in fours or 
tetrads. 

In cover-glass preparations from the meningeal exudate 
the diplococcus frequently is situated inside leucocytes, and 
sometimes within the nucleus (Fig. 54). The appearances 
are very much like those of gonorrheal pus. It is decolor- 
ized by Gram's method. 

Blood-serum. — The colonies appear after about twenty-four 
hours, and after forty-eight hours may attain a diameter of 



28o PATHOLOGICAL TECHNIQUE. 

2 or 3 mm. They are round, colorless, shining, slightly 
convex or flat, moist, and viscid-looking. They may be- 
come confluent. 

Agar-agar. — Round, flat, grayish, translucent, moist, shin- 
ing colonies, attaining a diameter of 2 or 3 mm. after twenty- 
four hours in the incubator. They may become confluent, 
and in a "slant" culture the growth appears as a grayish, 
translucent, moist, shining streak about 3 mm. in width, 






Fig. 54. — Diplococcus intracellularis meningitidis in polynuclear leucocytes of 
meningeal exudate (Jaeger). 

with smooth margins. Under a low magnifying power 
the colonies are homogeneous, semi-translucent, and not 
granular. 

Sugar-agar Stab-culture. — Feeble growth not extending 
all along the line of inoculation. 

Bouillon. — Medium slightly to moderately clouded. At 
the bottom of the tube a whitish sediment, which may rise 
as a viscid string when the tube is shaken. 

Potato. — Very feeble or doubtful growth, giving the sur- 
face of the potato a moist appearance at the most. 



PATHOGENIC BACTERIA AND FUNGI. 28 1 

Litmus-milk. — Growth without visible change in the 
medium. 

Gelatin. — Feeble growth. No liquefaction. 

Vitality. — The organism quickly dies out under cultiva- 
tion. It seems to survive somewhat better on blood-serum 
than on agar-agar, but cultures on the former only seventy- 
two hours old may be found no longer capable of growth 
after transplantation. 

Pathogenesis. — Intraperitoneal inoculation of guinea-pigs 
and rabbits gives very uncertain results. Mice are said to 
succumb to subcutaneous inoculation, with some invasion of 
the blood by the organism. 




Fig. 55. — Diplococcus intracellularis meningitidis in leucocytes. Cover- 
glass preparation from peritoneal exudate in a guinea-pig; X 2000 (Wright 
and Brown). 

Exceptionally, we have found that the intraperitoneal in- 
jection of a bouillon suspension of a twenty-four-hour 
blood-serum culture in the quantity of about 1 c.c. would 
kill guinea-pigs within forty-eight hours. 

At the autopsy there is an accumulation of a cloudy or 
blood-stained fluid in the peritoneal cavity, some little en- 
largement of the spleen, and some injection of the perito- 



282 PATHOLOGICAL TECHNIQUE. 

neum. Microscopical examination of the exudate shows 
numerous leucocytes crowded with the diplococci (Fig. 55). 
The culture-test gives no evidence of general infection of 
the blood. 

Occurrence. — Found in the meningeal exudate of epidemic 
cerebrospinal meningitis. It is situated mainly inside the 
pus-cells, some of which may contain many diplococci. In 
some cases the presence of the organism in the exudate may 
be difficult or impossible to demonstrate, and it is probable 
that it rapidly dies out. It has been found in the arthritis 
and in the pneumonia which sometimes accompany the 
disease, and in the nasal secretion both of sick and of well 
individuals. A general invasion of the circulation by the 
micro-organism may be shown by cultures from the blood, 
but not in the majority of cases. 

Diagnosis (see also section on Lumbar Puncture). — In 
exudates suspected of containing it, cover-glass preparations 
should be stained by the method for gonococci (see page 276). 
The presence of micrococci, often in the pus-cells, decolor- 
ized by Gram's method, is sufficient for its identification in 
the meningeal exudate as far as our present knowledge goes. 

Cultures, preferably on blood-agar plates or 1 per cent, 
glucose-agar plates, may be positive when smear prepara- 
tions are negative. They should be made also from the 
secretion of the nose and nasopharnyx. From isolated, 
suspicious colonies composed of Gram-negative cocci resem- 
bling the meningococcus transplants should be made to 
bouillon containing 1 per cent, glucose and 1 per cent, 
calcium carbonate. With the bouillon cultures so obtained 
agglutination tests with immune serum should be carried 
out in dilutions of 1 : 100, 1 : 200, 1 : 400. Mixtures of 
serum dilutions and bouillon culture of 0.5 c.c. each are 
placed in small tubes and incubated at 55 C. for twenty- 
four hours. 

Bacillus of Influenza. — Morphology. — Very small ba- 
cilli, with rounded ends and of variable length, sometimes 
growing into long forms, more or less bent or curved (see 

Fig- 55). 

Stains more deeply at the ends than in the middle, and in 
the long forms shows irregularity of staining. The faintly 



PATHOGENIC BACTERIA AND FUNGI. 



283 



stained areas are very sharply defined, as in the case of 
the typhoid bacillus. 



£ m 


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♦ :•• 


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Fig. 



56. — Influenza bacilli from a culture on blood-agar ; X 2000 (Wright and 
Brown). 




Fig. 57. — Bacilli of influenza in a leucocyte in a cover-glass preparation from 
sputum. A pneumococcus also in the same leucocyte and other pneumococci 
free. The small size of the bacillus of influenza will be apparent by comparison 
with the pneumococci ; x 2000 (Wright and Brown). 

In cover-glass preparations from bronchial secretions (see 
Fig. 56) the bacillus appears smaller and less plump than 



284 PATHOLOGICAL TECHNIQUE. 

it does in preparations from cultures. It also does not show 
irregularities in staining. 

Cultivation. — Does not grow in the ordinary culture-media, 
but maybe cultivated on agar- agar " slants," the surfaces 
of which have been smeared with a few drops of sterile 
blood. The blood of man, rabbits, guinea-pigs, pigeons, or 
frogs will serve for this purpose, the best growth being 
obtained with pigeon's blood. The blood may be ob- 
tained from a needle-prick, and spread over the surface 







^1 



Fig. 58. — Bacillus of influenza; colonies on blood agar (F. T. Lord; photo, by 

L. S. Brown). 

of the agar-agar by means of the platinum loop. The skin 
should be previously thoroughly washed with alcohol and 
ether, and the first drops of blood should not be used. 
Human blood is best obtained from the lobe of the ear 
or from the finger. Tubes thus prepared are only rarely 
contaminated. 

Colonies. — After twenty-four hours in the incubator the 
colonies appear as minute colorless, glassy, transparent 
points resembling small drops of dew. They never attain 
any size, and do not become confluent. They are barely 



PATHOGENIC BACTERIA AND FUNGI. 285 

visible to the unpractised eye, and require a low magnifying 
power to be seen clearly. Under the low magnifying power 
they are translucent, homogeneous, not ' granular, and cir- 
cular in outline (Fig. 58). 

Decolorized by Gram's method. Not motile. Will not 
grow without oxygen. 

Pathogenesis. — The ordinary laboratory animals are not 
susceptible to infection with this organism. 

Occurrence. — Found in the exudate of the respiratory tract 
in influenza, frequently inside of leucocytes (Fig. 57). It 
may be present in the small bronchi and in the exudate of 
broncho-pneumonia in this disease. It has been observed in 
purulent meningitis secondary to influenza. 

F. T. Lord, working in the Laboratory of the Massachusetts 
General Hospital, found influenza bacilli in 60 of 100 unselected 
specimens of sputa repeatedly negative for tubercle bacilli. In 29 
of these 60 cases the bacilli were present in great numbers. 
Eleven cases were of acute and 1 8 of chronic inflammation of the 
respiratory tract. In the chronic cases he demonstrated the per- 
sistence of influenza bacilli in the sputum for months or years. 

Lord believes that influenza bacilli are very commonly present 
in sputa apart from epidemics of influenza, and that chronic infec- 
tion with influenza bacilli is not infrequently mistaken clinically 
for tuberculosis. 

Diagnosis. — Microscopical examination of cover-glass 
preparations of the bronchial sputum shows very small, 
short, round-ended bacilli, often in very large numbers and 
frequently in the pus-cells. These bacilli frequently occur 
in pairs and resemble pairs of cocci. Their ends may be 
more deeply stained than the central portions. For the 
staining of cover-glass preparations of the sputum PfeifTer 
recommends that a very dilute carbol-fuchsin solution be 
applied for five to ten minutes. The cover-glass preparation 
is to be made from a distinctly purulent portion of the 
sputum. Staining with Loffler's methylene-blue solution 
also gives good results. See also W. H. Smith's method 
for staining the capsule of the pneumococcus, page 237. 

The bacillus of influenza may be cultivated from the 
sputum by breaking up a small portion of a distinctly puru- 
lent character in 1 or 2 c.c. of bouillon, and then spreading 



286 PATHOLOGICAL TECHNIQUE. 

a platinum loopful of the suspension over the surface of a 
blood-agar-agar slant, which is then placed in the incubator. 
After eighteen to twenty-four hours the characteristic col- 
onies may be visible with the aid of a hand-lens. These 
should not grow in ordinary media unless blood or hemo- 
globin be present, and should have the morphology of the 
bacillus of influenza. 

F. T. Lord obtains the best results by using as a culture -medium 
i part sterile horse-blood and 2 parts nutrient agar-agar in 
"slant" tubes. The blood is mixed with fluid agar -agar at 40 
C. The colonies of the influenza bacilli may attain a diameter 
of more than a millimeter on this medium. The horse-blood may 
be easily obtained from any antitoxin plant and may be kept on 
hand in test-tubes for months without impairment of its utility for 
cultural purposes. 

To Stain the Influenza Bacillus in Sections. — 1. Harden in 
alcohol. 

2. Stain half an hour or more in carbol-fuchsin diluted 
with 20 parts of water. 

3. Wash out in a watch-glass of water to which is added 
a drop of glacial acetic acid until the section appears gray- 
violet in color. 

4. Alcohol, xylol, balsam. 

For Zenker's fixed tissues the eosin-methylene-blue stain 
is recommended. 

Bacillus Pertussis (Bordet-Gengou).— The bacillus 
of whooping-cough was discovered by Bordet and Gengou 
in 1900 in the sputum of acute cases of the disease. The 
first cultures were obtained by them in 1906. 

Morphology.— -The micro-organism is an extremely small 
non-motile bacillus. Frequently it is so short that it re- 
sembles a micrococcus. It is more regularly ovoid and some- 
what larger than the influenza bacillus, and shows less tend- 
ency to pleomorphism and involution. It stains readily'with 
the ordinary aniline dyes, and usually more intensely at the 
ends than in the middle. It is decolorized by Gram's 
method. 

Cultivation. — It is difficult to start a growth of the whoop- 
ing-cough bacillus, although the organism grows readily 
enough after it has become accustomed to an artificial 



PATHOGENIC BACTERIA AND FUNGI. 



287 



medium. Bordet and Gengou obtained their first cultures 
on a special potato-blood-agar medium which they devised 



*m 



W* ^ 



m • 



» t » 



?f Si 



Fig. 59. — Bacillus pertussis from a culture ; X 2000 (Mallory). 




Fig. 60. — Bacillus pertussis in masses between the cilia of the epithelial cells 
lining the trachea ; X about 2000 (Mallory). 



and which remains to-day the only medium on which a 
growth can be started. 



288 PATHOLOGICAL TECHNIQUE. 

The medium is prepared as follows : 

(a) A glycerin extract of potato is prepared by steaming 
in an autoclave ioo grams of sliced potato in 200 c.c. of a 
4 percent, solution of glycerin in water. 

{b) To 50 c.c. of this extract add 1 50 c.c. of a 0.6 per cent, 
salt solution and 5 grams of agar. Melt the mixture in the 
autoclave, pour 2 to 3 c.c. into test-tubes, and sterilize. 

(c) After sterilization add an equal volume of sterile defi- 
brinated rabbit blood, or, preferably, human blood, mix the 
two fluids, slant the tubes, and solidify the mixture. 

Cultures should be made from the sputum during the early 
days of the disease. The organisms grow slowly at first, so 
that the colonies are barely visible after twenty-four hours. 
After forty-eight hours they are plainly visible as small, 
grayish, rather thick colonies. Later generations grow with 
luxuriance. After they have been cultivated for some time 
on the potato-blood-agar medium they will grow readily on 
plain blood-agar or ascitic-agar and in ascitic broth, or broth 
to which blood has been added. 

Occurrence.— -The bacillus of whooping-cough is found in 
large numbers and in practically pure culture in the early 
stages of the infection. It occurs free in the secretion, and 
also within polymorphonuclear leucocytes. Study of post- 
mortem cases has shown that the organism is located be- 
tween the cilia of the epithelial cells lining the trachea and 
bronchi. This location seems to be characteristic for this 
organism in man. 

In experimental work with this bacillus on animals, es- 
pecially puppies and young rabbits, it is necessary to bear in 
mind that the Bacillus bronchisepticus, the cause of distem- 
per, snuffles, etc., in animals, is often present, and that it is 
of the same size and occupies the same peculiar position be- 
tween the cilia of the cells lining the air-passages, including 
the nasal cavity. It differs from the bacillus of whooping- 
cough in being motile and in producing alkali in litmus-milk. 

Bacillus Coli Communis. — Synonyms: Bacterium coli 
commune; Colon bacillus. 

Morphology. — A medium-sized bacillus with rounded ends, 
often short or even coccus-like, but may grow in long forms. 



PATHOGENIC BACTERIA AND FUNGI. 



289 



Faintly staining, sharply defined areas are present in the pro- 
toplasm (Fig. 61). 

Decolorized by Gram's method. 







''0' v-; ** 


^ / 


V 


*• / 


3 • 7 ' 


4 

t 


'/ ' S . v> 


/" 



Pig. 61. — Bacilli coli communis from a bouillon culture, showing the irregu- 
larity of staining of the bacillus ; X 2 °°° (Wright and Brown). 




Fig. 62. — Bacillus coli communis: superficial colony two days old upon a 
gelatin plate ; X 21 (Heim). 

Motility. — Usually not motile, but some varieties show 
sluggish independent movement. 

Flagella may be demonstrated by the special methods 

19 



29O PATHOLOGICAL TECHNIQUE. 

of staining. They are less numerous than in the case of the 
typhoid bacillus. 

Blood-serum. — Rounded, grayish-white, slightly elevated, 
viscid-looking colonies, which may attain a diameter of 
3 mm. after twenty-four hours in the incubator. 

Gelatin Slant. — Grayish translucent strip with wavy mar- 
gins. The gelatin is not liquefied. Growth is more rapid 
than in the case of the typhoid bacillus. 

A single colony on a gelatin plate in shown in Fig. 62. 

Glucose- gelatin Stab. — Growth along the line of stab in 
the form of confluent spherical colonies, and on the surface 
about the point of entrance of the needle as a thin gray 
circular layer. Gas-bubbles are produced in the gelatin 
from fermentation of the glucose. The gelatin is not 
liquefied. 

Glucose-agar-agar Stab. — Growth essentially the same as 
in the preceding, except that the gas-formation is more 
marked. 

Litmus-milk. — Turned pink and usually coagulated. 

Potato. — Dirty grayish or brownish, viscid-looking layer. 

Dunham's Peptone Solution. — Marked indol-production. 
This is shown by the appearance of a red color in the cul- 
ture after the addition of 5 drops of concentrated sulphuric 
acid, c. p., and 1 c.c. of a 1 : 10,000 solution of sodium 
nitrite. The culture in the peptone solution should have 
been at least twenty-four hours — or, better, forty-eight hours 
— in the incubator before the test is made. 

Bouillon. — Markedly clouded, with formation of a sedi- 
ment. The clouding is more marked than in the case of 
the typhoid bacillus. 

Lactose-litmus-agar-agar Slant. — Growth has a pink color, 
and the blue color of the medium is changed to red. Gas 
is produced. 

Action on Other Sugars. — Acid and gas are produced in 
media containing maltose and mannite, but not in media 
containing saccharose. 

Pathogenesis. — "Its virulence as tested upon animals is 
variable, but is generally manifest only after inoculation of 



PATHOGENIC BACTERIA AND FUNGI. 29 1 

large doses, which kill by intoxication rather than infection " 
(Welch). 

The lesions produced are not sufficiently characteristic to 
be detailed here. 

Occurrence. — Occurs constantly in the intestinal canal, and 
is widely distributed in the external world. 

" The colon bacillus is a frequent invader of the internal 
organs in all sorts of diseases, especially when there are in- 
testinal lesions. It manifests no evident pathogenic action 
in most of these cases, and is then without clinical signifi- 
cance. It occurs frequently associated with other bacteria 
in infected wounds and other inflammations of exposed sur- 
faces. Here also it does not usually appear to cause serious 
disturbance. The fact that the colon bacillus is so common 
and widely distributed, and found so often as a harmless in- 
vader, should lead to much caution in interpreting the sig- 
nificance of its presence when it occurs in definite lesions. 
There is no doubt, however, that it may be pathogenic for 
man. It plays an important role in inflammations of the 
urinary tract and biliary passages ; also, but usually with 
less independence, in peritonitis and appendicitis. 

" The list of diseases in which it may be found is a very 
long one, and includes inflammations in all organs and parts 
of the body. In general its pathogenic properties are of a 
mild character. One of its leading roles is to invade terri- 
tory already occupied by other bacteria or previously 
damaged. It may be concerned in the production of gall- 
stones, in the interior of which it has been found by the 
writer with great frequency " (Welch, Dennis's System of 
Surgery ', vol. i.). 

The bacillus above described is to be regarded as a type of a 
group of bacilli constituting the so-called "colon group" of 
bacilli. These present certain quantitative differences among 
themselves which are not quite sufficient to characterize them as 
distinct species. 

Welch regards as belonging to this group the Bacillus pyogenes 
feetidus, distinguished by the stinking odor of its cultures, and 
the Bacillus lactis aerogenes, which is characterized chiefly by its 
plumper form, its more energetic gas-production, its rapid coagu- 
lation in milk, and its denser growth in cultures. 



292 PATHOLOGICAL TECHNIQUE. 

Theobald Smith suggests that only those forms may be re- 
garded as typical members of the group which grow on gelatin 
in the form of delicate bluish or more opaque whitish expansions 
with irregular margin, which are actively motile when examined 
in the hanging-drop from young surface colonies taken from gela- 
tin plates, which coagulate milk within a few days ; grow upon 
potato either as a rich pale or brownish-yellow deposit, or merely 
as a glistening, barely recognizable layer, and which give a dis- 
tinct indol-reaction. Their behavior in the fermentation - tube * 
must conform to the following scheme : 

x The fermentation-tube is a special form of culture-tube which may be 
obtained from dealers in bacteriological supplies. The closed branch of the 
tube should be completely filled with culture-fluid, but no more fluid should 
be placed in the tube than can be conveniently held by the open branch of the 
tube, so that if gas be formed in the closed branch the culture-fluid will not be 
forced out of the apparatus. The bubbles which collect at the top of the closed 
branch, after heating during sterilization, should be removed by an appropriate 
tilting of the tube. Theobald Smith, who was the first to demonstrate the 
great value of the fermentation-tube in bacteriology, thus describes the mode 
of its use : 

" The tubes are kept, after inoculation, in the thermostat at 37 C. A mark 
made on the sides of the closed branch at the end of every twenty-four hours 
with a glass pencil furnishes an approximate record of the rate of gas-produc- 
tion. Unless this is done it is impossible to know precisely when the formation 
of gas is at an end, and also whether or not the volume of gas has been dimin- 
ished by absorption. It is best to wait four or five days after the production 
has ceased before making a final examination. This is done by noting the 
condition of the growth, the reaction of the fluid in the bulb,* and the maxi- 
mum quantity of gas produced. This is most easily done by laying directly 
on the tube a glass millimeter rule, and noting the tube length occupied by gas. 
The entire length of the closed branch is also noted, making due allowance for 
the upper convex extremity and the lower constriction. This mode of meas- 
urement is sufficient, since only comparative values are desired. For the same 
reason all barometric and thermometric corrections are omitted in these approxi- 
mate estimations. 

" The examination of the gas produced was limited to the determination of 
the quantity of carbon dioxid and of the explosive character of the gas remain- 
ing after the absorption of C0 2 by sodium hydrate. These facts are deter- 
mined by the following simple manipulations : 

" The bulb is completely filled with a 2 per cent, solution of NaHO, and 
closed tightly with the thumb. The fluid is shaken thoroughly with the gas, 
and allowed to flow back and forth from bulb to closed branch and the reverse 
several times, to insure intimate contact of the C0 2 with the alkali. Lastly, 
before removing the thumb, all the gas is allowed to collect in the closed branch 

* The reaction was noted by placing a drop of the fluid on delicate litmus- 
paper. The cultures were occasionally boiled to drive off any CO2. In no 
case did the reaction with the litmus-paper change. 



PATHOGENIC BACTERIA AND FUNGI. 293 

Variety a. One per cent, dextrose-bouillon (at 37 C). 

Total gas, approximately 1/2 ; H/C0 2 approximately 2/1 ; 

reaction strongly acid. 
One per cent, lactose bouillon : 
As in dextrose-bouillon (with slight variation). 
One per cent, saccharose-bouillon : 

Gas-production slower than in the preceding, lasting from 
seven to fourteen days. Total gas finally about 2/3 ; 
H/C0 2 nearly 3/2. The final reaction in the bulb may 
be slightly acid or alkaline, according to the rate of gas- 
production. 
Variety /?. The same in all respects excepting as to its behavior in 
saccharose-bouillon. Neither gas nor acids are formed in it. 




Fig. 63. — Typhoid bacilli from a bouillon culture, showing characteristic irregu- 
larity in staining and variability in length ; X 2000 (Wright and Brown). 

Bacillus of Typhoid Fever. — Synonyms: Bacillus 
typhi abdominalis ; Bacillus typhosus; Typhoid bacillus. 

Morphology. — Medium-sized bacilli with rounded ends, 
generally short (Fig. 63), but sometimes long or thread-like, 

so that none may escape when the thumb is removed. If C0 2 was present, a 
partial vacuum in the closed branch causes the fluid to rise suddenly when the 
thumb is removed. After allowing the layer of foam to subside somewhat, 
the glass scale is again applied to the closed branch, and the amount of C0 2 
absorbed may thus be measured. In all cultures of this character thus far ex- 
amined the gas remaining was explosive in character, and probably hydrogen. 
The explosive character of this residue is easily demonstrated as follows : The 
cotton plug is replaced, and the gas in the closed branch allowed to flow into 
the bulb, and mix with the air there present. The plug is then removed, and 
a lighted match inserted into the mouth of the bulb. The intensity of the 
explosion varies with quantity of air present in the bulb." 



294 



PA THO L O GICA L TE CHNIQ UE. 



and frequently showing faintly stained, sharply defined areas 
in their protoplasm (Figs. 63 and 64). 

Decolorized by Gram's method. Does not form spores. 



■*» 




**> 


t • 


1 


♦ I 


V' % 






§ 1 * 




* 



Fig. 64. — Typhoid bacilli from a culture on potato, showing unstained areas in 
the bacilli and polar granules ; X 2000 (Wright and Brown). 

Motility. — Very marked. 

Flagella (Fig. 66) may be demonstrated by the special 
methods of staining described elsewhere. 




Fig. 65. — Bacillus of typhoid fever: superficial colony two days old, as seen 
upon the surface of a gelatin plate ; x 20 (Heim). 

Blood-serum. — Round, grayish, viscid-looking colonies, 
which may attain a diameter of 2 mm. after forty-eight 
hours in the incubator. 



1 



PATHOGENIC BACTERIA AND FUNGI 



295 



Bouillon. — Clouded, with the formation of some sediment. 
The clouding of the medium is not so marked as in the case 
of the bacillus coli communis. In general, the growth of the 
typhoid organism is not so vigorous on culture-media as is 
the growth of the bacillus coli communis. 




Fig. 66. — Typhoid bacilli, from a culture on agar-agar, showing flagella, from a 
preparation stained by Dr. Hugh Williams; X 2000 (Wright and Brown). 



When to a bouillon culture a small quantity of the blood- 
serum of a typhoid-fever patient is added, the bacilli lose 
their motility and aggregate in clumps (' 'serum reaction"). 

Gelatin Slant. — Broad translucent streak with wavy, 
irregular margins. The gelatin is not liquefied. Growth is 
slower than that of the bacillus coli communis in the same 
medium. 



296 PATHOLOGICAL TECHNIQUE. 

An isolated colony, slightly magnified, on gelatin, is 
shown in Fig. 65. 

Glucose-gelatin Stab. — Growth all along the line of inocu- 
lation in the form of confluent spherical grayish colonies, and 
on the surface about the point of entrance of the platinum 
wire in the form of a circular translucent grayish layer. No 
production of gas-bubbles. No liquefaction. 

Glucose Agar-agar. — Growth similar to that in the pre- 
ceding. No gas-formation. 

Litmus-milk. — No visible change. 

Potato. — Growth occurs, but it is usually invisible. 

Dunham's Peptone Solution. — No indol-production — i. e. 
no red color appearing in the twenty-four- to forty-eight- 
hour cultures after the addition of 5 drops of concentrated 
sulphuric acid, c. p., and 1 cubic centimeter of a solution of 
sodium nitrite, 1 : 10,000. 

Action on Sugars. — In media containing glucose, maltose, 
and mannite acid is produced, but no gas. Neither is 
produced in media containing lactose or saccharose. 

Pathogenesis. — The inoculation of animals is usually with- 
out results if moderate quantities of the organism are used. 
Sometimes, however, death occurs apparently from the 
effects of the toxic material injected. 

Occurrence. — Found in the spleen in large numbers at 
autopsies in typhoid fever. Its presence may also be demon- 
strated in the intestinal lesions, rose spots, mesenteric lymph- 
glands, liver, bile, kidneys, urine, and blood of the heart. 
As a rule, the number of bacilli found in the liver, kidneys, 
and blood of the heart is small. In the bile they may be 
numerous and may persist in it for a long period of time 
after the disease has subsided. In some cases the urine con- 
tains enormous numbers of the bacilli. 

The typhoid bacillus may also occur in the suppurative 
sequelae of typhoid fever, especially those involving bones. 
In these conditions, however, it may be accompanied by the 
pyogenic cocci. Occurs in contaminated water. 

Typhoid bacilli in stained sections are generally best 
hunted for with a low power. The characteristic colonies 
which they form are easily recognized. Good results in 



PATHOGENIC BACTERIA AND FUNGI. 



297 



staining can be obtained with Loffler's methylene-blue solu- 
tion used in the manner already described, but the stain is 
never very intense. For rendering the bacilli rather more 
prominent, so that small groups of them may be recognized, 
Flexner has advised the two following methods: 

A. — I. Stain paraffin sections in Loffler's methylene-blue 
solution for two hours. 

2. Acetic-acid solution, I : iooo, for several minutes. 

3. Dehydrate in absolute alcohol. 

4. Oil of cloves to clear and differentiate. 

5. Xylol, several changes. 

6. Xylol balsam. 

B. — 1. Stain sections in Stirling's gentian-violet solution 
for ten minutes. 

2. Acetic-acid solution, 1 : 1000, for some minutes. 

3. Dehydrate quickly in 95 per cent, alcohol. 

4. Transfer to slide, blot, add oil of cloves to clear, and 
differentiate. Change the oil several times until the desired 
differentiation is obtained. 

5. Wash off section several times with xylol. 

6. Xylol balsam. 

The Blood-serum Reaction in Typhoid Fever; Widal 
Reaction. — A few drops of the blood of a suspected case 
of typhoid fever are collected in a small test-tube, either 
from the finger or the ear. After clotting has taken place, 
transfer a drop of the serum by means of a medicine- 
dropper to forty drops of a recent bouillon culture of the 
typhoid bacillus. After mixing, place a drop of the mixture 
on a slide, cover it with a cover-glass, and examine with a 
high-power dry objective. 

If the bacilli are seen to be motionless and to be agglom- 
erated in clumps within twenty minutes, the reaction is to 
be regarded as present and typical. If the clumping occurs 
within thirty minutes, but free bacilli are still moving, the 
reaction is to be regarded as doubtful. The reaction is 
present, as a rule, only after the first week of typhoid fever. 
The slide and cover-glass should be sterilized in a flame 
after the test is completed. The bouillon culture used 
should be grown at room-temperature and should not be 



298 



PA THOLOGICAL TECHNIQUE. 



more than four or five days old. Before carrying out the 
test it is well to assure one's self that the bacteria are 
actively motile by examining a drop of the culture as above 
indicated. The stock cultures of the typhoid bacilli are 
best made on agar-agar. 

The reaction may also be obtained from the dried blood. 
A few drops of the blood may be collected on a glass slide 
or a piece of paper and allowed to dry. It may then be 
brought to the laboratory, where as much of the dried blood 
as would correspond to a drop is scraped from the slide into 
a small test-tube, containing forty drops of a bouillon-culture 



\ ' 




! 


••-, 






'"K ■ ' * 






;■/ <■■'■-■■'■ 




V 




® 


\ . ■ 









Fig. 67 — Showing the clumping of typhoid bacilli in the serum-reaction. 
Wet preparation, not stained. At one point a crenated red blood-corpuscle is 
seen (Wright and Brown). 

of the typhoid bacillus, or as much of the paper as may be 
assumed to contain one drop of blood is placed in forty drops 
of the bouillon culture and allowed to soak for a few minutes 
therein. Microscopical examination is then made with the 
mixture thus obtained as above indicated. 

Cultivation of the Typhoid Bacillus from the Blood 
during Life. — One method of doing this is indicated on 
page 425. It is important that the blood should be diluted 
with a large excess of sterile bouillon — say, 1 or 2 c.c. to 
100 or 200 c.c. of bouillon in flasks. 

The Ox-bile Method. — Test-tubes containing 5 c.c. of plain 
ox-bile sterilized by steam are used. If a precipitate appears 



■PATHOGENIC BACTERIA AND FUNGI. 299 

on heating, it may be filtered off before using without detri- 
ment to the medium. The blood of the patient is added to 
the bile in the test-tube up to 2.5 c.c. in amount. It may be 
obtained from one of the superficial veins of the forearm by 
means of a syringe, or by pricking the lobe of the ear with a 
lance-pointed knife and squeezing out drop by drop into the 
tube of bile. The mixture of bile and blood is then incubated 
at 37° C. for twelve to fifteen hours, when transfers of a few 
loopsful are made to other culture-media in order to pre- 
pare to establish the identity of the bacteria that may have 
developed. 

Paratyphoid Bacilli. — These bacilli have been found 
associated with inflammatory processes and with fevers clin- 
ically resembling typhoid fever. They differ from the typhoid 
bacillus chiefly in that they produce acid and gas in glucose, 
maltose, and mannite media and show different agglutination 
reactions. Two types of them, known as types "A" and 
"B," are generally recognized. Type A behaves in all 
other respects essentially like the typhoid bacillus. Type B 
does not coagulate milk, but makes it alkaline and, after 
ten days or more, translucent. Its colonies are generally 
larger, less translucent, and may have a porcelain white 
color. Its growth on potato may appear as a thick brown- 
ish layer. 

Differential Diagnosis between the Bacillus of Typhoid 
Fever and the Bacillus Coli Communis. — The most im- 
portant points of difference between these two organisms are 
as follows, and to distinguish with certainty between them it 
is necessary that attention be paid to all of them : 

Motility. — The typhoid bacillus is actively motile, the 
bacillus coli communis not motile or exceptionally motile. 

Potato Cultures. — The typhoid bacillus usually grows in- 
visibly, the bacillus coli communis as a dirty, slimy layer. 

Gas-production in Media Containing- Glucose. — The ba- 
cillus coli communis produces gas, the typhoid bacillus 
does not. 

Litmus-milk Cultures. — The bacillus coli communis changes 
the blue color of the medium to a pink color and usually 



300 PATHOLOGICAL TECHNIQUE. 

coagulates the milk. The typhoid bacillus produces no 
visible change. 

Indol-production. — The bacillus coli communis produces 
indol, the typhoid bacillus does not. 

Serum or Clump Reaction. — The typhoid bacillus shows 
the clump reaction, while the bacillus coli communis does 
not. As it is not always possible to have a typhoid serum 
at hand by which to determine whether this reaction is 
present, a stock of dried blood from a typhoid case, con- 
tained in filter-paper, may be kept ready for use. That this 
is quite practicable has been clearly shown by Dr. Mark W. 
Richardson. The blood may be obtained from the heart at 
the autopsy of a typhoid-fever case by soaking a piece of 
filter-paper with it. This is allowed to dry, and then is cut 
into pieces about I cm. square. When it is desired to make 
the test, one of these pieces is extracted with ten or fifteen 
drops of water, and a drop of this extract is mixed with a 
drop of an eighteen- to twenty-four-hour bouillon culture 
on a slide, covered with a cover-glass, and examined with 
the high-power dry lens. Dr. Richardson has found that 
the blood under these conditions retains for months its 
" clumping " power with reference to the typhoid bacillus. 

Other differences are — the production of a red color in 
litmus-lactose agar-agar by the bacillus coli communis, and 
no change in color of this medium by the typhoid bacillus, 
and the slower and less vigorous growth of the typhoid 
bacillus in culture-media. 

Bacillus Dysenterise (Shiga). — This bacillus resembles 
the typhoid bacillus in morphology, but in general it is 
plumper and less frequently appears in filamentous forms. 
Involution forms quickly develop in glucose-agar cultures. 
It is decolorized by Gram's method, and does not form 
spores. It is not motile. The bacillus grows in bouillon 
and on agar and gelatin, both in plate and tube cultures, 
with appearances very similar to those of the typhoid 
bacillus. It does not produce gas in media containing 
glucose or other sugars. 

Typical examples of the bacillus do not produce indol 



PATHOGENIC BACTERIA AND FUNGI. 301 

in peptone solution, but some strains have been found to 
do so. 

Litmus Milk. — During the first two or three days the milk 
becomes a pink color, but later becomes blue. It is never 
coagulated. 

Potato. — Growth, at first in the form of a moist, colorless, 
slimy, almost invisible layer, becoming, after two or three 
days in the incubator, of a yellowish to brownish tint with 
discoloration of the potato. 

Mannite Litmus Agar. — This medium consists of ordinary 
nutrient agar-agar containing 1 per cent, of mannite and I 
per cent, of a 5 per cent, aqueous solution of litmus. The 
agar-agar should have been made up with meat infusion 
free from muscle-sugar (see p. 193). In stab cultures on 
this medium the typical bacillus decolorizes the agar in the 
depths, but near the surface the original blue color remains 
unchanged. Some strains of the bacillus, however, change 
the color of the upper layers to red, as do typhoid and colon 
bacilli. Thus two varieties of the bacillus may be distin- 
guished with reference to their effect on this culture-medium. 
Both of these varieties have been isolated from the same 
dysenteric stools. A third variety is distinguished by pro- 
ducing acid in maltose media. All three varieties do this 
in glucose media. None produces acid from lactose or 
saccharose. 

The bacillus exhibits the agglutination reaction with the 
serum of dysenteric cases and with the serum of animals 
immunized against the bacillus. 

Pathogenesis. — The bacillus is pathogenic for the usual 
laboratory animals, especially for mice and guinea-pigs 
which may die in from twenty-four to forty-eight hours after 
subcutaneous or intraperitoneal inoculation. At the autopsy 
there may be found local inflammation at the seat of inocu- 
lation, ecchymoses of the serous membranes, serous or sero- 
hemorrhagic exudate in the pleural or peritoneal cavities, 
enlargement of the spleen, and hyperemia or hemorrhage in 
the intestinal walls. 

Occurrence. — In the stools, intestinal contents, and in the 
ulcerated mucous membrane of acute dysentery, whether 



302 PATHOLOGICAL TECHNIQUE. 

sporadic or epidemic. It may be found in the mesenteric 
lymphatic glands, but is not found in the blood, in the 
spleen, or in other viscera. 

Examination of Feces for Typhoid, Paratyphoid, 
and Dysentery Bacilli. — The examination should be 
made immediately; typhoid bacilli disappear in more than 
10 per cent, of the specimens of feces in twenty- four hours; 
paratyphoid organisms do not degenerate so rapidly. 

Make a suspension of the feces in broth to about the 
viscosity of thin cream and allow the coarser particles ten 
to twenty minutes to settle. On a plate of brilliant green 
(three to five per million) agar place two loops, on an Endo 
plate one loop of the suspension. With sterile smearing rod 
(glass or platinum) spread the material consecutively on the 
surfaces of a series of plates (two to five of each medium) 
in order to get isolated colonies on one or more of the plates 
after incubation at 37 C. for eighteen to twenty- four 
hours. For purposes of comparison control plates of the 
media with known strains of the bacilli in pure culture 
should always be made. 

Fish characteristic colonies, inoculate a tube of Russell 
medium from each colony studied. If the reactions are 
characteristic of typhoid, paratyphoid, or dysentery bacilli, 
suspensions in broth should be made and the motility and 
agglutination in known serum tested after a few hours' 
incubation, the sera having been carefully titrated for group 
agglutinins and to determine the limits of reaction with 
homologous strains. Determine morphology and staining 
reaction. 

Preparation of Media. — Brilliant Green Agar Plates. — 
3 c.c. of Andrade indicator (which has been prepared by 
mixing 100 c.c. of 0.5 per cent, aqueous acid fuchsin and 
16 c.cc. n/i NaOH solution an hour before use) is added 
to 100 c.c. of melted sterile beef extract; \\ per cent, agar 
reacting plus 0.2 acid to phenolphthalein and containing 
1 per cent, lactose and 0.1 per cent, dextrose, n/i HC1, or 
n/i NaOH is added if necessary to give a faint pink color 
when the medium is cold. Test a sample. Just before 
pouring plates add 0.3 to 0.5 c.c. of a 0.1 per cent, 
aqueous solution of brilliant green. Do not heat after 



PATHOGENIC BACTERIA AND FUNGI. 303 

adding the brilliant green. For the examination of fresh 
specimens three plates will suffice, otherwise five will give 
better results. 

Note. — The action of the brilliant green dye on pure cultures of the ba- 
cilli should be accurately standardized. The dye Bayer No. 574, now in 
use, almost completely inhibits the colon bacillus when 0.3 c.c. of the 0.1 per 
cent, aqueous solution is added to 100 c.c. of the agar medium, but it does 
not affect the growth of the typhoid bacillus materially. 

The solution of the dye is prepared by washing 0.1 gram from a watch- 
glass on the scales with 100 c.c. of boiling water. The solution will keep a 
month or rjiore, but should be restandardized with each new batch of agar. 

Endo-agar Plates. — To 100 c.c. of sterile melted 3 per 
cent, agar containing 1 per cent, lactose and reacting plus 
0.4 (acid to phenolphthalein) 1 c.c. of fuchsin indicator is 
added, thoroughly mixed, and the plates poured. The 
fuchsin indicator is prepared by adding 1 c.c. of a saturated 
alcoholic solution of basic fuchsin to 10 c.c. of a 10 per 
cent, aqueous solution of anhydrous sodium sulphite. 

Russell Tube Medium. — (See note below.) 

To the ordinary beef extract i| per cent, agar, 1 per cent, 
lactose, 0.1 per cent, dextrose, and 3 per cent. Andrade 
indicator are added and the medium sterilized in live steam 
(Arnold ioo° C.) twenty minutes on three successive days; 
then slanted. In the modified Russell medium, maltose is 
substituted for lactose. This is used in the identification of 
the dysentery bacilli. Recent investigation has shown that 
the Russell medium modified by the addition of 1 per cent, sac- 
charose offers certain advantages, especially when isolating 
members of the paratyphosus group. A considerable percent- 
age of non-lactose fermenting organisms which produce gas 
in dextrose also produce acid on the slant by fermenting the 
saccharose. This reaction separates them from the typhoid, 
paratyphoid, and dysentery groups which do not produce 
acid (red) slants on this medium. 

The agar is inoculated on the surface and by deep punc- 
ture. 

Note. — This medium should not be sterilized at higher temperatures or 
longer exposures than above indicated. A small amount of the medium 
should be cooled. If the reaction is correct the cooled medium will have a 
slight pink color. If necessary n/i NCI or n/i NaOH must be added to the 
medium until this result is obtained. 



304 PATHOLOGICAL TECHNIQUE. 

Bacillus Proteus (Proteus Vulgaris). — Morphology. — 
Bacilli of very variable length, sometimes appearing like 
cocci or as filaments. 

Motile, being provided with terminal flagella. Does not 
stain by Gram's method. 

Colonies in Gelatin. — Rapid growth with liquefaction of 
the gelatin. In a medium containing 5 per cent., instead of 
10 per cent., of gelatin prolongations from the margins of 
the colonies may be formed. These may become separated 
from the mother colonies and form daughter colonies. 
Motions may be observed in these prolongations. 

Gelatin Stab. — Rapid liquefaction along the line of inocu- 
lation with cloudiness of the liquefied gelatin and a floccu- 
lent deposit. 

Agar-agar Slant. — Widely spreading, thin, moist, grayish- 
white layer. 

Potato, — Dirty white, moist layer. 

Litmus-milk. — Turned pink and slowly coagulated. 

Odor. — The cultures generally have a- putrefactive odor. 

Pathogenesis. — Intravenous, intraperitoneal, or intramus- 
cular inoculations of rabbits may produce death in twenty- 
four to thirty-six hours after moderately large doses. Liq- 
uefied gelatin-cultures are said to be more virulent than 
bouillon cultures. Guinea-pigs seem to be less susceptible 
than rabbits to infection with this organism. 

Occurrence. — This bacillus and its varieties are among the 
most common and widely distributed putrefactive bacteria. 
It occurs in the intestinal contents. In pathological exam- 
inations it may be found in peritonitis and in abscesses, 
usually associated with other bacteria. It may also invade 
the circulating blood. 

The so-called " proteus group" includes several varieties of 
similar organisms — viz. the proteus vulgaris, the proteus mirabilis, 
and the proteus Zenkeri. The latter does not liquefy the gelatin, 
while the proteus mirabilis liquefies it slowly. 

Bacillus Diphtherise. — Morphology. — Bacilli varying 
markedly in size and shape, of irregular outline, and show- 
ing great variability in the staining of different parts of 



PATHOGENIC BACTERIA AND FUNGI. 



305 






• 



1 



Fig. 68. — Diphtheria bacilli from a culture on blood-serum, stained by 
Loffler's methylene-blue solution, showing deeply stained points; X 200 ° 
(Wright and Brown). 



- 








1 *> 




* A < 




V " 






4 — t- " 




KV1 


-*>L^>V * ^ 






/■ ^ **sr v. 


\ 

1 


■ 






1 


rK^m^/ &* •/• 






^j^f r- 1 






^ \i v 






4/1 / r* pj* 




%A* 1 



Fig. 69. — Diphtheria bacilli from a culture on blood-serum, stained by 
Loffler's methylene-blue solution, showing long and irregularly shaped forms of 
the bacillus, as well as the irregularity of staining ; X 2000 (Wright and Brown). 
20 



306 



PA THOLOGICAL TECHNIQUE. 



their protoplasm (Figs. 68, 69). The presence in a palely 
tinted rod of deeply stained granules and points, frequently 
situated at the extremities, and the occurrence of irregular 
forms, often club-like in shape, with a constriction in the 
middle, are appearances which are very characteristic of 
the bacillus when grown upon blood-serum and stained 
with Loffler's methylene-blue solu- 
tion. Its morphology and staining 
peculiarities are so characteristic 
when cultivated upon blood-serum 
that the microscopical examination 
is in most cases sufficient for its iden- 
tification. When grown upon other 
culture - media than blood - serum, 
however, its morphology and stain- 
ing peculiarities are not so charac- 
teristic, and they may vary markedly 
in different media. 

Stained by Gram's method. Not 
motile. 

Blood-serum. — Round, elevated, 
smooth colonies of the color of the 
medium. They may attain a diam- 
eter of 2 mm. after forty-eight hours 
in the incubator. 

Bouillon. — Grows usually in the 
form of fine grains at the bottom of 
the tube and adherent to the sides, 
the bouillon remaining clear or be- 
coming slightly clouded. The reac- 
tion of the media rapidly becomes 
acid, but changes to alkaline after 
a variable length of time. 

Potato. — Growth not visible to the naked eye. The 
bacillus grows, however, to a certain extent, and usually as- 
sumes very atypical and irregular forms (involution forms). 
Agar -agar and Gelatin. — The growth on these, media is 
slower and more feeble than upon blood-serum (Fig. 70). 
It presents nothing remarkable. 




Fig. 70. — Bacillus diph- 
therias; agar-agar culture 
(photograph by Dr. Henry 
Koplik). 



PATHOGENIC BACTERIA AND EUNGI 307 

Pathogenesis. — Subcutaneous inoculations of guinea-pigs 
are fatal in thirty-six to seventy-two hours in the case of 
virulent cultures. The lesions produced consist usually of 
edema, hemorrhage, and fibrino-purulent exudation about 
the point of inoculation in the subcutaneous tissue, hemor- 
rhagic enlargement of the lymphatic glands, congestion and 
edema of the lungs, hemorrhages into the suprarenal capsules, 
and less frequently necrosis of the liver and pleural effusions. 
Histological examination of the lymph-glands shows marked 
"fragmentation" of the nuclei of the cells, giving rise to 
numerous deeply staining globules of chromatin scattered 
throughout them. The bacilli are ordinarily found only 
about the point of inoculation, but cultures from the various 
organs will sometimes show the presence of the bacilli m 
some of them. 

Toxin- production. — The effects produced by infection with trie 
bacillus diphtherial are due to the action of a so-called toxalbumin 
or " toxin " which the organism manufactures in its growth. The 
poisonous substance is produced in cultures. Its presence may be 
demonstrated by inoculating an animal with a small quantity of 
the filtrate, obtained by passing a bouillon culture some weeks old 
through an unglazed porcelain filtering apparatus, by which all 
the bacteria are removed from the fluid. 

The " toxin" is contained in solution in the filtrate. If this 
be fairly rich in "toxin," the injection of -^ c.c. subcutane- 
ously into a guinea-pig should lead to the death of the animal in 
three or four days with the various lesions above described. The 
local reaction, however, is not so marked as in the case of inocu- 
lation with the bacilli. With the ordinary bouillon the produc- 
tion of a great amount of "toxin " by the growth of the diph- 
theria bacilli in it is very uncertain. Theobald Smith has recently 
shown that this uncertainty is due to the presence of variable 
amounts of muscle-sugar from the meat used in the preparation 
of the bouillon, and that this substance prevents the accumulation 
of toxin. He has found that that bouillon yields the most toxin 
which has the least muscle-sugar in it. He prepares such bouillon 
as follows : " Beef infusion, prepared either by extracting in the 
cold or at 6o° C, is inoculated in the evening with a rich fluid 
culture of some acid-producing bacterium (I use temporarily 
B. coli) and placed in the thermostat. Early next morning the 
infusion, covered with a thin layer of broth, is boiled, filtered^ 
pepton and salt added, and the neutralization and sterilization 
carried on as usual." This bouillon is placed in two 500 c.c. 
Erlenmeyer flasks, 250 c.c. in each flask. In these, cultures are 
made and kept for at least eight days in the incubator. After 



308 '" ■Pathological technique. 

this time a fair amount of toxin may be assumed to have 
developed, and the contents of the flask are then filtered 
through a porcelain cylinder. A filtrate is to be regarded as 
containing a reasonable amount of toxin if T L c.c, injected 
subcutaneously, kills a medium-sized guinea-pig in three days. 
The filtrate containing the '" toxin " can be preserved by the 
addition of 0.5 per cent, pure carbolic acid. 

Occurrence.— -The bacillus diphtheriae occurs in the local 
lesions in all cases of true diphtheria, in rhinitis fibrinosa, 
and in many .cases of the milder forms of acute inflammation 
of the air-passages. It may persist in the mucous mem- 
brane of the throat and nose long after convalescence has 
teen established. 

..In fatal cases of diphtheria the organism is nearly always 
present in the lungs, and it may be often found by culture- 
methods more or less generally distributed in compara- 
tively small numbers throughout the internal organs. In 
the majority of djphtheria autopsies an invasion of the blood- 
stream by the: streptococcus pyogenes, and sometimes by 
other bacteria, maybe demonstrated by cultures. The ba- 
qillus may also be found in company with other bacteria in 
ulcerated or excoriated surfaces on the skin, as well as in 
other suppurative processes, in individuals affected with 
diphtheria, and on the soiled linen of the patient. The in- 
fection of WQunds-with the bacillus diphtheriae has also been 
observed without coincident diphtheria. 

Diagnosis.— Y\\z bacteriological diagnosis of infection 
with the bacillus diphtheriae depends upon the characteristic 
morphology and peculiarities of staining, as well as rapidity 
of growth, which this organism presents when cultivated 
upon coagulated blood-serum. The identification by direct 
Gover-glass examination of the exudate is very uncertain. 

The method is as follows : A blood-serum culture-tube is 
inoculated with a small amount of the material from the 
mucous membrane affected, and is placed in the incubator 
twelve to eighteen hours. After this length of time the re- 
sulting growth is examined by cover-glass preparations 
stained either with Loffler's methylene-blue solution or by 
one of the special methods given below. 



PATHOGENIC BACTERIA AND FUNGI. 309 

The bacillus diphtherise, if present, may then be recog- 
nized and differentiated from other bacteria present in the 
preparation by its characteristic morphology and peculiarity 
of staining, described on page 304. The gross appearances 
of the culture present little that is characteristic, as a rule*, 
and the main reliance is to be placed on the microscopic ex- 
amination. Early in the infection the greater part of the 
growth may be made up of the specific bacilli, but toward 
convalescence they fall into the minority. The ordinary 
forms of agar-agar culture are not suitable for use in the 
bacteriological diagnosis of diphtheria, owing to the com- 
parative feebleness of the growth of the organism on these 
media, and because of the fact that its microscopic appear- 
ances when cultivated on such media are not sufficiently 
characteristic. 

The material for culture is very conveniently obtained by 
means of sterilized cotton swabs. In collecting this material 
the swab is removed from its test-tube and touched to the 
affected areas of the mucous membrane of either the nose or 
throat. It is then to be gently rubbed over the surface of a 
blood-serum culture-tube, or it may be replaced in the test- 
tube and the inoculation of the culture-tube made later in 
the laboratory. In the latter case the inoculation should be 
made within an hour or two after the material has been col- 
lected, the infected swab meanwhile being prevented from 
drying by firmly replacing the cotton plug. 

In cases with membrane-formation the greatest number 
of bacilli are on the surface or in the upper layer of the 
membrane, and the swab should therefore be touched to 
these portions rather than to the tissue beneath. 

Special Methods of Staining" the Bacillus Diphtherise.— 

Owing to the fact that the bacillus diphtheriae may be recognized 
by its peculiar morphology and characteristic staining in cover- 
glass preparations from its growth upon certain culture-media, as 
already pointed out, various special staining methods have been 
devised for accentuating and rendering more striking to the eye 
the peculiar deeply stained points and granules in the bodies of 
the individual bacilli, which have been referred to as of great im.- 
portance in the identification of the organism. 



3IO PATHOLOGICAL TECHNIQUE. 

These special methods of staining are said to be of great advan- 
tage in cases where only a few specific bacilli may be suspected 
to be present among a large number of other bacteria. 

Neisser' s Method. — i. Stain for one to three seconds in a solu- 
tion which is made as follows : i gram of methylene-blue (Grue- 
bier), in powder, is dissolved in 20 c.c. of 96 per cent, alcohol. 
To this add 950 c.c. of distilled water and 50 c.c. of glacial acetic 
acid, and filter. 

2. Wash in water. 

3. Stain for three to five seconds in a solution of vesuvin 
(Bismarck brown), made by dissolving 2 grams of the dye (in 
powder) in 1000 c.c. of boiling distilled water. 

4. Wash in water, and mount. 

The diphtheria bacilli stained by this method appear as pale 
brown rods bearing bluish-black granules, usually of oval shape 
and of a diameter somewhat greater than the rod. The majority 



* ■ . • * • 









•- • -♦♦*.. 






•• < 



Fig. 71. — Diphtheria bacilli from blood-serum culture stained according to 
Neisser's method ; x 2000 (Wright and Brown). 

of the bacilli show a granule at each end or at only one end, but 
not rarely three granules are present, one being near the middle of 
the rod. More granules than these are exceptional (see Fig. 71). 
The bacilli must have been grown on Loftier' s blood-serum 
medium, coagulated at ioo° C. , and the culture must be at least 
nine hours and not more than twenty-four hours old. 



PATHOGENIC BACTERIA AND FUNGI. 



31 



Hunt's Method. — 1. Stain in saturated aqueous solution of 
methylene-blue one minute without heating. 

2. Wash in water. 

3. Cover with aqueous solution of tannic acid, 10 per cent., for 
ten seconds. 

4. Wash in water. 

5. Stain in saturated aqueous solution of methyl-orange one 
minute, without heating. 

6. Wash in water. 

7. Dry, and mount in balsam. 

By this method the granules, etc., are dark blue or almost black, 
and stand out very sharply against the light-green coloring of the 
body of the bacillus (see Fig. 72). The solution of methyl-orange 
should be freshly prepared, for it deteriorates in a few days. 



4>f 



/.~ 






\i 



._$• 



fWL 



Km 



-T 



Jit ^ 



Fig. 72. — Diphtheria bacilli from blood-serum culture stained according to Hunt's 
method ; X 2000 (Wright and Brown). 

Mallory's Stain for the Diphtheria Bacillus. — This stain- 
ing method was devised because Loffler's solution made up 
with some of the methylene-blues now in the market stains 
poorly and does not keep well. Extended use of this solution 
shows that it keeps perfectly and can be used repeatedly. 



Methylene-blue, 

Water, 

Glacial acetic acid, 



1 gm.; 
100 c.c; 
3 c.c. 



Stain smear preparations 15 to 30 minutes; wash off with 
water and dry. Overstaining does not occur. The polar 
bodies or granules are stained intensely. The body of the 



312 PATHOLOGICAL TECHNIQUE. 

bacterium stains lightly. The solution cannot be recom- 
mended as a general stain for bacteria because it is not 
intense enough, unless structures similar to polar bodies 
are present. 

Scarlet Fever. 1 — Mair has recently described a diplo- 
coccus scarlatinas, an oat-shaped coccus, as a possible cause 
of the disease. Mallory and Medlar have independently 
described a bacillus scarlatinas. The two organisms seem 
to resemble each other closely both morphologically and 
culturally and may prove, on further study, to be identical. 
Both produce sloughs of the skin in animals when inocu- 
lated subcutaneously. 

The description given here is of bacillus scarlatinas. 

Bacillus Scarlatina. — A bacillus approaching more nearly 
the strepto-pneumococcus group than the diphtheria group. 

Morphology. — A bacillus slightly smaller than the diph- 
theria bacillus, which varies from coccoid to long bacillary 
forms. It is generally broadest in the central portion and 
tapers slightly toward the ends, which are rounded. In 
smears from growth on solid media the organisms are ar- 
ranged in masses similar to diphtheria bacilli. Pairs and 
short chains are quite common. Long chains, ten to thirty 
organisms, are very common in the water of condensation 
and in fluid media. 

Stains readily with ordinary aniline dyes ; strongly Gram- 
positive; non-motile. There is some irregularity of stain, 
more marked in older cultures, but no polar bodies have 
been observed. 

Cultures. — Grows best, at least at first, under anaerobic 
conditions. 

Serum-agar. — Under anaerobic conditions the organism 
produces a colony 0.5 to 1 mm. in diameter in eighteen to 
twenty-four hours. The colony is round and has an entire 
or slightly undulating edge. It is convex, opalescent, with a 
refractile central portion in the larger and older colonies. 
Under the microscope a finely granular structure is seen 

x Mair, W.: "Experimental Scarlet Fever in the Monkey," Jour. Path, 
and Bact., 1915, xix, 443-445; "On the Etiology of Scarlet Fever," Jour. 
Path, and Bact., 1916, xx, 366-383. Mallory and Medlar: "The Etiology 
of Scarlet Fever," Jour. Med. Research, 1916, xxxv, 209-229. 



PATHOGENIC BACTERIA AND EUNGI 



313 





Fig. 73. — Scarlet fever — Gram-Weigert stain: a, Erosion on surface of pharynx, 
lined with masses of bacilli; considerable fibrin in inflammatory reaction; X 200; 
b, smear preparation from a similar erosion in pharynx of a patient showing the 
characteristic morphology of the bacillus of scarlet fever; X 1000; c, masses of ba- 
cilli in upper surface of membrane in larynx; X 200. 



throughout. The colonies tend to remain discrete, although 
in thickly seeded plants they become confluent. 



314 PATHOLOGICAL TECHNIQUE. 

Dunham's Peptone Solution. — Growth is slight; indol is 
not produced. 

Potato. — Thin colorless growth occurs. 

Gelatine. — Growth moderate; slight liquefaction takes 
place. 

Bood-serum. — Good colorless growth. 

Litmus Milk. — Red; coagulated. 

Pathogenesis. — Injection of large doses subcutaneously 
produces local loss of hair and sloughing of the skin. Mair 
found his organism moderately pathogenic for mice, but 
considerably less so than diplococcus pneumoniae. 

Occurrence. — The primary essential lesion of scarlet fever 
consists of necroses of the surface epithelium in the upper 
respiratory tract, beginning perhaps usually in or on the 
tonsils. The necroses may be followed by membrane for- 
mation or erosion, or by a combination of the two. In 
severe cases of infection the process may extend to the 
larynx, trachea, bronchi and lungs, and also into the esopha- 
gus. Early in the disease (twenty-four to forty-eight hours 
after the appearance of the skin eruption) the bacillus 
scarlatinae is found in large numbers in the upper part of 
the fibrinous membrane and lining the erosions; also in the 
bronchi and in the alveoli of the lungs. 

Bacillus of Glanders (Bacillus Mallei). — Morph- 
ology. — Bacilli of medium size, variable in length, having 
round or conical ends, and frequently showing faintly 
stained areas in their protoplasm (Fig. 74). The larger 
forms of the bacillus are usually slightly bent or wavy in 
outline. Slight irregularities in shape may be observed. 
The morphology varies considerably on different culture- 
media. 

In cover-glass preparations from the lesions the bacilli 
usually appear somewhat longer and thicker than the tuber- 
cle bacillus, and show numerous sharply defined, unstained 
or faintly stained areas in their protoplasm (Fig. 75). They 
have rounded or conical ends, and are sometimes slightly 
irregular in shape. As a rule, they are present in small 
numbers. If Loffler's methylene-blue solution is used for 
staining the cover-glass, it should be heated ; if carbol- 



PATHOGENIC BACTERIA AND FUNGI 



315 



fuchsin is used, it should be followed by a slight decolori- 
zation with 95 per cent, alcohol to better differentiate the 
bacilli. Gram-negative; non-motile. 

Blood-serum. — Rounded, elevated, colorless, viscid-looking 
colonies, growing slowly and becoming well developed after 




Fig. 74. — Glanders bacilli from a young culture on potato ; X 2000 (Wright and 

Brown). 




Fig. 75. — Glanders bacilli in a cover-glass preparation from a lesion in a 
guinea-pig, showing the marked irregularity in the staining of the bacilli ; X 2000 
(Wright and Brown). 

thirty-six hours in the incubator. They may attain a diam- 
eter of 2 or 3 mm., and after a time they assume a brown- 
ish tint. 

Potato. — After thirty-six hours in the incubator a rather 
thick, colorless, viscid-looking layer appears, which soon 
assumes a brownish tint and resembles honey in appearance. 



316 PATHOLOGICAL TECHNIQUE. 

Later the brown color changes to a dark reddish-brown, 
and the growth becomes thicker and more opaque, while 
the potato takes on a dark gray color. 

Bouillon. — Diffusely clouded, with the formation of a 
viscid sediment. 

Litmus-milk. — Gradually turned red and coagulated. 

Agar-agar and Gelatin. — Growth not especially charac- 
teristic. 

Pathogenesis. — When inoculated subcutaneously into 
guinea-pigs, the characteristic results are swelling and in- 
flammation of the scrotum, appearing after a variable num- 
ber of days, often after a week. 

The animals usually survive several weeks, with ulceration 
at the point of inoculation. The lesions produced consist 
in suppurative processes or abscess-formations in or about 
the testes, in the lymph-glands, in the anterior nares, about 
the joints, and in other situations, besides small grayish 
nodules or areas in the viscera — the so-called " glanders 
tubercles." The suprarenal capsules usually show red 
areas, and they may be enlarged. On microscopical exami- 
nation the small nodules as well as the extensive suppura- 
tive areas will be found to be composed of necrotic material 
containing leucocytes and fragments of chromatin. The 
distribution and extent of the lesions vary with each animal, 
but the involvement of the testis or its membranes is prac- 
tically constant and pathognomonic of the bacillus of glan- 
ders. This involvement of the testis may consist, in early 
cases, in the presence of yellow foci in or about the tunica 
vaginalis, or in later cases the organ may show large yellow 
areas with purulent softening. 

Intraperitoneal inoculation with virulent cultures may be 
followed by death within forty-eight hours, with fibrinous 
exudate on the peritoneum in which minute grayish nodules 
are seen. The nodules are made up of a material which is 
apparently mainly dead or degenerated leucocytes and des- 
quamated peritoneal endothelium, together with many chro- 
matin fragments. 

In these acute cases also microscopical examination of 
the spleen and liver may show the presence of small nodules 



PATHOGENIC BACTERIA AND FUNGI. 317 

identical in structure with those seen in the more chronic 
cases. For the purpose of producing with cultures the 
characteristic lesions of the testis or its coverings it is better 
to inoculate the animal subcutaneously, for in the rapidly 
fatal intraperitoneal inoculations with virulent cultures these 
may not show any marked changes. 

The bacilli may be cultivated from the lesions, but not 
from the blood of the heart, in the chronic cases. They 
may be present in the blood of the heart, however, in small 
numbers in rapidly fatal infections following intraperitoneal 
inoculation. 

Field-mice may die from subcutaneous inoculation in 
about seventy -two hours. The most conspicuous lesion pro- 
duced is enlargement of the spleen, with the presence in it of 
minute grayish nodules. White mice are immune. Rabbits 
are not so susceptible as guinea-pigs to the infection. 

Occurrence. — Found in the lesions of glanders and of 
farcy, and may invade the blood-stream in small numbers 
in acute cases of infection. Grows in the tissues in clumps 
or groups as well as scattered. In lesions on exposed sur- 
faces it may be accompanied by the pyogenic cocci. We 
have succeeded in demonstrating the presence of the bacillus 
in the sputum of a case of human glanders by inoculation 
of a guinea-pig with the sputum. 

Diagnosis. — In a case of suspected glanders the dis- 
charges from sinuses or ulcerated surfaces, or the contents 
of pustules, are to be examined for the presence of the 
bacillus of glanders by the usual methods. 

The material for examination may be collected on "swabs." 
With this a guinea-pig is to be inoculated and cultures and 
cover-glass preparations are made. If the material be from 
sinuses or ulcerated surfaces, the isolation of the bacillus by 
cultures will be difficult, owing to the presence of other or- 
ganisms. The guinea-pig is to be inoculated in the peri- 
toneal cavity by introducing the infected swab into it through 
an incision in the abdominal wall, or by injecting about 1 
c.c. of a suspension in bouillon of the suspected material 
into the peritoneal cavity with a hypodermic syringe. 

If the bacillus of glanders is present, the scrotum will 



318 PATHOLOGICAL TECHNIQUE. 

usually show the characteristic swelling and inflammation in 
the course of three or four days, and death will occur after 
some weeks. In some cases the animal may die in thirty- 
six or forty-eight hours. In any case the characteristic 
lesions of glanders will be found as described elsewhere, 
and the bacillus may be isolated from them by cultures. 
The spleen will practically always yield glanders bacilli in 
pure culture even if no macroscopical lesion can be made 
out. 

In cultures the organism should show those character- 
istics of morphology, of culture, and of pathogenesis which 
have been described above. 

To Stain the Glanders Bacillus in Sections. — The bacilli 
are usually not numerous, and are scattered about in 
a mass of deeply staining fragmented nuclei, so that often 
they are recognized with great difficulty. The ordinary 
eosin-methylene-blue stain, after fixation in Zenker's fluid, 
can be highly recommended for demonstrating them. The 
following special methods have long been used for the same 
purpose. 

Libffler's Methylene -blue Stain for Sections. — i. Stain 
paraffin sections twenty minutes in Loffler's methylene-blue 
solution or in equal parts of aniline-methyl-violet and 
i : 10,000 KOH solution. 

2. Place for five seconds in the following solution : 

Distilled water, 10 c.c. ; 

Concentrated sulphuric acid, 2 drops; 

5 per cent, oxalic acid, I drop. 

3. Wash out quickly in distilled water. 

4. Absolute alcohol. 

5. Xylol. 

6. Xylol balsam. 

It is recommended to place the section for a few minutes 
before staining in the 1 : 10,000 caustic-potash solution. 

Schutz's Method. — 1. Stain twenty-four hours in equal 
parts of concentrated alcoholic solution of methylene-blue 
and caustic potash, 1 : 10,000. 



PATHOGENIC BACTERIA AND FUNGI. 



319 



2. Wash in acidified water. 

3. 50 per cent, alcohol for five minutes. 

4. Absolute alcohol for five minutes. 

5. Xylol. 

6. Xylol balsam. 

Noniewicz's Method. — 1. Stain in Loffler's methylene- 
blue solution two to five minutes. 

2. Wash in water. 

3. Decolorize one to five seconds in 

\ per cent, acetic acid, 75 parts ; 

\ per cent, aqueous solution of tropeolin, 25 " 

4. Wash in water. 

5. Dehydrate section on slide with filter-paper; then in 
the air ; finally, over small flame. 

6. Clear by dropping xylol on it repeatedly. 

7. Xylol balsam. 




Fig. 76. — Bacillus of chancroid in smear preparation from pus (Lincoln Davis ; 
photo by L. S. Brown). 

Bacillus of Chancroid (Bacillus of Ducrey). — In 

smears from the lesions the bacilli appear as short, round- 
ended rods, about 1,5 p. long and 0.5 fi thick, occurring 
characteristically but not always in chains. The middle 
portion of the rods does not stain so deeply as the ends. 
The bacilli are decolorized by Gram's method of staining, 
and are not motile. The following description of the cul- 



320 



PA THOL GICAL TECHNIQ UE. 



tural peculiarities of the chancroid bacillus are based on the 
observations of Dr. Lincoln Davis in the Laboratory of the 
Massachusetts General Hospital. 

The bacillus does not grow on the ordinary culture- 
media, but may be cultivated in blood or in media contain- 
ing one-third its volume of blood. It is essential that the 
blood be fresh. In tubes containing blood, or a mixture of 
bouillon and blood, after twenty-four hours in the incubator, 
the growth appears as whitish flocculi at the bottom of the 







Fig. 77. — Bacillus of chancroid from culture (Lincoln Davis ; photo by 
L. S. Brown). 

tube. These flocculi are composed of tangled chains of the 
bacilli, the chains being often of extreme length. The 
individual bacilli, as a rule, have the same morphology and 
staining reactions as in the smears from lesions, but occa- 
sionally long or even filamentous forms may be seen. On 
the surface of slant tubes, composed of a mixture of fresh 
blood and agar, the bacillus forms, after forty-eight hours in 
the incubator, small, rounded, grayish colonies difficult to 
pick up with the platinum wire, because they tend to glide 
before it. The bacilli from these colonies appear in smear 
preparations in short chains and singly. Involution forms 
are early apparent among the bacilli in all cultures. The 



PATHOGENIC BACTERIA AXD FUNGI 32 1 

bacillus dies out in cultures after about three days. Upon 
a monkey of the genus Macaccus, small ulcerations in the 
skin were produced by inoculation with cultures. 

Method of Isolation from the Lesions. — Dr. Davis found 
that pure cultures were readily obtained by inoculating a 
small quantity of freshly drawn human blood in small tubes 
with material from the lesions, the fresh blood apparently 
inhibiting or destroying other bacteria. These small tubes 
containing blood are most easily prepared in a way devised 
by J. H. Wright. A small glass tube, about 5 or 6 cm. long 
and 4 or 5 mm. in internal diameter, is drawn out into a fine 
caliber at one end, and is then sterilized throughout in the 
gas-flame. When cool, the pointed extremity of the tube is 
immersed in the blood obtained from a needle-prick in the 
skin of the dorsum of the thumb near the nail, and then by 
manipulation of the tube the blood is caused to flow into it. 
In this way a sufficient quantity — say, 0.2 to 0.5 c.c. — is 
easily collected in the tube, after which the pointed end is 
sealed in the flame and the tube is ready to be inoculated. 
The other end of the tube is plugged with cotton, which is 
impregnated with paraffin to prevent evaporation. The 
skin, before being pricked, is sufficiently cleansed by soap 
and water, followed by alcohol. A small tourniquet is 
applied about the base of the thumb, to increase the flow of 
blood from the needle-prick. 

Bacillus Pyocyaneus (Bacillus of Green Pus). — 
Morphology. — Small bacilli with rounded ends (Fig. 78). 

Decolorized by Gram's method (Welch). Motile, and is 
provided with a flagellum at one end. Does not form 
spores. 

Blood-serum colonies grow rapidly, are not especially 
characteristic in form, and liquefy the medium, imparting to 
it a dark greenish color. 

Gelatin Stab. — Liquefaction in funnel form, with green 
fluorescence of the upper portions of the medium. The 
liquefied gelatin is densely clouded, and there may be a 
viscid pellicle on the surface. 

Agar-agar Stab. — A green fluorescence in the upper layers 
of the medium, which later becomes a dark blue-green. 
21 



322 



PA THOL O GICA L TE CHNIQ UE. 



Potato. — Slightly elevated, brownish, viscid layer. The 
potato in some cases assumes a green color, in others a 
brown color. In some cultures the potato when touched 
with the platinum wire takes on a green color at the point 
touched. This is the so-called " chameleon phenomenon," 
and it is best observed in cultures several days old. 

Bouillon. — The growth is in the form of flocculi and a 
delicate surface pellicle. The fluid acquires a green color. 

Litmus-milk. — Acid reaction with coagulation. 

Dunham' s Pepton Solution. — Indol is produced. 

Colonies on Gelatin Plates (Fig. 79). — Development is rapid. 
Young colonies are provided with a fringe of delicate fila- 
ments about their periphery. As growth progresses and 
liquefaction becomes more advanced, the central mass of the 




Fig. 78. — Bacillus pyocyaneus showing flagella, from a preparation stained by 
Dr. Hugh Williams ; X 2000 (Wright and Brown). 

colony sinks into the liquefied depression, while at the same 
time there is an extension of the colony laterally. . . . At this 
stage the colony, when slightly magnified, may present vari- 
ous appearances, the most common being that shown in Fig. 
79. The gelatin between the growing colonies takes on a 
bright yellowish-green color, but, as growth is comparatively 
rapid, it is quickly entirely liquefied, and one often sees the 
colonies floating about in the pale-green fluid. 

Pathogenesis. — Subcutaneous inoculation of guinea-pigs 
and rabbits with I c.c. of a virulent bouillon culture may 
produce purulent infiltration and inflammatory edema of the 
tissue about the point of inoculation, and death may follow 
in eighteen to thirty-six hours. Intraperitoneal inoculation 
may result in a sero-fibrinous or purulent peritonitis with 






PATHOGENIC BACTERIA AND FUNGI. 



3 2 3 



fatal result. In fatal inoculations the bacillus is found in the 
various viscera, but not in great numbers. Animals inocu- 
lated with small amounts may survive with merely local 
lesions, and an immunity may be produced. 

Several varieties of this bacillus have been described, but their 
differences do not seem to be of sufficient importance to justify 
their separation into distinct species. 




( -'- / ; ■[■'■\i:y^f^^& 



v 



/'~-.^ 




Fig. 79. — Bacillus pyocyaneus : colonies upon gelatin (Abbott). 

Occurrence. — " Is widely distributed, occurring often on the 
human skin, in the feces, and outside of the body. In wounds, 
stains the dressings bluish-green and produces a somewhat 
characteristic offensive odor. 

" Increases suppuration of wounds, usually with little con- 
stitutional disturbance. Is found not infrequently in perfora- 
tive peritonitis and appendicitis, sometimes in phlegmons, 
otitis media, broncho-pneumonia, and inflammation of serous 
membranes, associated usually with other bacteria. 

" It was found by H. C. Ernst in tuberculous pericarditis. 
Often found in diarrheal and dysenteric discharges. May 
cause general infection in human beings. With or without 
general infection it may cause hemorrhagic and necrotic en- 
teritis, a form of pyocyaneous infection in human beings which 
we have repeatedly observed at autopsy. Instances of in- 
vasion of the body from wounds by the bacillus pyocyaneus 
have not been observed " (Welch). 

Bacillus of Bubonic Plague. — Morphology. — In the 



3 2 f \. PATHOLOGICAL TECHNIQUE. 

tissues the organism occurs as a medium-sized short bacillus 
with rounded /ends. In cultures its size and length vary 
and its median portion may be swollen so that an ovoid 
form is produced; it may grow in pairs and in chains, 
and it may occur as long, thread-like forms. Involution 
forms of elliptical or round shape, and often of large size, 
sometimes resembling yeast-cells, are frequent in old cult- 
ures or in cultures on special media. These involution 
forms are easily produced by cultivation on agar-agar con- 
taining 2y 2 to 3^ per cent, of sodium chlorid. 




Fig. 80. — Bacillus of bubonic plague (Yersin). 

• Staining.— -The organism stains with the usual aniline dyes, 
and is decolorized by Gram's method of staining. In the 
tissues it stains more deeply at its extremities than at its 
central portions, and it sometimes appears to possess a cap- 
sule. The polar staining may sometimes be brought out 
in cultures by weak staining solutions or by decolorization 
by alcohol. It is not motile, and it does not form spores. 

• Gelatin Plates. — The colonies on the surface appear after 
twenty-four to forty-eight hours at 22° C. They are flat, 
rOund, and white or yellowish white in color. Under a low 
magnifying power the central portion of the colony is gran- 
ular, while the marginal portion is clear. The colonies do 
not spread over the surface of the medium. 

Gelatin Stab. — Growth all along the line of inoculation 



PATHOGENIC BACTERIA AND FUNGI. 32$ 

with the formation of a layer of growth at the surface of a 
whitish color. There is no liquefaction of the gelatin. 

Gelatin Slants. — A whitish or slightly yellowish layer 
presenting nothing characteristic. 

Agar-agar Plates. — The colonies on the surface appear 
first as dew-drops, and have already attained their maximum 
development after twenty-four to forty-eight hours in the 




01 



Fig. 81. — Bacilli of plague and phagocytes, from human lymphatic gland; X 800 

(Aoyama). 

incubator. They will then grow white in color, and present 
an opalescent or iridescent margin. Under the microscope 
they are distinctly granular. Considerable difference in size 
may be observed among the colonies. The larger colonies 
are said to be less virulent for animals than the smaller 
colonies, and it is claimed that these larger colonies when 
transplanted give rise to large colonies again. 

Agar-agar Slant. — The colonies tend to become conflu 
ent, and the growth is somewhat viscid. 

Bouillon. — The fluid usually remains clear, and the growth 
appears in the form of a granular or flocculent sediment 
which may here and there adhere to the wall of the tube t 
In bouillon cultures richly inoculated and retained in a per- 
fectly and undisturbed position at room-temperature for 



326 PATHOLOGICAL TECHNIQUE. 

some days a characteristic appearance is produced. In 
twenty-four to forty-eight hours islands of growth appear 
underneath the surface in the form of flakes. In the next 
twenty-four to forty-eight hours there grow down into the 
fluid from the flakes long, stalactite-like masses, the liquid 
remaining clear. In four to six days the islands of growth 
have become more compact and solidified. If the flask be 
now slightly disturbed, the islands fall to the bottom, bring- 
ing with them the stalactite-like growths. The latter are 
very fragile. In addition to these appearances there is a 
deposit of growth on the wall of the flask and at the bottom, 
as well as a ring of growth on the margin of the surface of 
the liquid. 

Milk. — Growth without coagulation. 

No production of indol. 

In neutral litmus bouillon the blue color is changed to red. 

There is no odor, and no pigment production. 

The organism is aerobic. 

It remains alive in cultures for five to six weeks at least. 

Growth occurs at all temperatures from 4 C. to 37 C. 
The best temperature for growth is 30 to 3 2° C. 

Pathogenesis. — The organism is pathogenic for a great 
variety of animals, including mice, rats, guinea-pigs, and 
rabbits. In these animals death generally follows in from 
two to six days after subcutaneous inoculation. The lesions 
produced are hemorrhagic edema at the seat of inoculation, 
enlargement of the lymphatic glands with more or less 
hemorrhage, enlargement of the spleen and its follicles. 
The bacilli are present in large numbers in the enlarged 
lymphatic glands and in the internal organs ; they are less 
numerous in the blood. Rats and certain other animals 
may be infected by feeding. Pigeons, chickens, and cattle 
are immune. 

Occurrence. — The bacillus is found in large numbers in 
the buboes, pustules, pulmonary lesions, and other localized 
lesions of the bubonic plague. It also may be found in 
larger of smaller numbers in the blood and internal organs 
generally, and it may be present in the sputum, bile, and 



PATHOGENIC BACTERIA AND FUNGI. 32 \J 

aivine discharges. The pus of the buboes which break 
spontaneously may be sterile. The organism may be 
demonstrated in the circulating blood of cases of plague. 

Bacteriological Diagnosis. — In cases of suspected plague 
the bacillus is to be sought for in the blood and in the 
buboes. In cases of pneumonia the sputum especially is 
to be examined. In the examination cultures as well as 
cover-glass preparations are to be used. 

Perhaps the most certain method of identification of the 
bacillus is the inoculation of the mucous membrane of the 
nose of the rat. The simple rubbing of a portion of the 
culture upon the mucous membrane appears to be sufficient 
to produce a fatal result in the rat if the culture is that of 
the genuine bacillus. As a culture-medium agar-agar or 
blood-serum is to be used in cases where there is no mixed 
infection. If there is mixed infection of the material to be 
examined, gelatin surface-cultures are to be made. 

The inoculation of animals for diagnostic purposes should 
be made with the greatest precaution to prevent the spread 
of the disease. 

Bacillus of Anthrax. — Morphology. — The organism 
grows in long segmented threads, the segments varying in 
length, but usually being two or three times as long as broad 
and having square or slightly concave ends. These seg- 
ments represent the bacillus, which is among the largest of 
the bacteria (Fig. 82). 

Pathogenesis. — Mice, guinea-pigs, and rabbits inoculated 
subcutaneously die with a general invasion of the blood by 
the organism. Mice are most susceptible to the infection, 
dying in about twenty-four hours, while guinea-pigs and 
rabbits survive longer. 

In all these animals the most striking lesion is a large soft 
spleen, and in the guinea-pig also an extensive inflammatory 
edema of the subcutaneous tissues. On microscopic exam- 
ination the bacilli will be found in the organs and blood of 
the heart. If the animal has been dead some time, the 
number of bacilli present in these situations will be very 
great, owing to the post-mortem growth. It is characteristic 



328 



PA THOLOGICAL TECHNIQ UE. 



of the bacillus of anthrax in cover-slip preparations from in- 
fected tissues that it should have a narrow capsule (Fig. 83). 




Fig. 82. — Bacillus of anthrax : portion of a colony three days old upon a gelatin 
plate; X 1000 (Frankel and Pfeiffer). 







x 







Fig. 83. — Bacillus of anthrax from spleen of a mouse (L. Frothingham). 



and show square or slightly concave ends. The capsule is 
not present in cultures. 



PATHOGENIC BACTERIA AND FUNGI. 



329 



Stained by Gram's method. Not motile. 

Forms oval spores in the middle of the short segments or 



» v 



£x 



si I 






•** 



* « m 

.J 



Fig. 84— Bacillus of anthrax, stained to show the spores ; X 1000 (Frankel and 

Pfeiffer). 

rods. The spores may be seen in blood-serum cultures after 
forty-eight hours in the incubator (Fig. 84). 




Fig. 85. — Bacillus of anthrax : gelatin stab-culture seven days old (Gunther). 

Blood-serum. — Irregularly rounded colonies, several milli- 
meters in diameter after twenty-four hours in the incubator. 
The colonies are grayish, finely granular, and have the ap- 



330 



PATHOLOGICAL TECHNIQUE. 



pearance of being made up of a dense network of delicate 
fibrillar. The blood-serum is slowly liquefied. 

Gelatin Stab. — Growth along the line of stab, with radiat- 
ing filaments extending laterally into the gelatin, which is 
slowly liquefied in funnel form (Fig. 85). 

Bouillon. — Growth in the form of cotton-like flakes and 
filamentous masses. No clouding of the medium. 

Agar-agar. — Matted network of translucent filaments. 



/ 



/ 



Fig. 86.— Bacillus of an- 
thrax ; cover-slip prepara- 
tion from vesicle. Stained 
by W. H. Smith's method ; 
X 1800 (W. H. Smith; 
photo, by L. S. Brown). 




Fig. 87. — Colony of bacillus of anthrax, 
slightly magnified (Fliigge). 



Under a lower magnifying power the growth is seen to be 
made up of twisted and contorted masses of filaments, giv- 
ing the appearance of curled hair (Fig. 87). 

Potato.— Grayish-white, rather thick, dry layer, having the 
appearance of frosted glass. 

Occurrence.— In malignant pustules, wool-sorter's disease, 
and intestinal anthrax. Found in the blood of animals dead 
of anthrax. In man the infection is usually localized at 
first at the point of inoculation, either on the skin or on the 
mucous membrane of the air-passages or intestinal tract. 
Later, a general invasion of the blood may occur and a fatal 
septicemia result. The organism or its spores may be pres- 
ent in wool or hides, and infection may take place from these. 
Diagnosis. — The bacilli may be found by the cover-glass 



PATHOGENIC BACTERIA AND FUNGI. 331 

examination of the contents of the small blebs and vesicles. 
The bacillus of anthrax may be identified by its morphology 
(see p. 327), its special characteristics being its large size and 
its square or concave extremities. 

The inoculation of a mouse at the root of the tail with some 
of the material from the pustule, and the production of the 
characteristic fatal septicemia, will render the identification 
certain. 

Bacillus MUCOSUS Capsulatus. — Morphology. — Bacilli 
of moderate size, usually two or three times as long as 
broad, with rounded ends, occurring frequently in pairs and 
sometimes in long forms. Occasionally in cultures it shows 




Fig. 88. — Bacillus mucosus capsulatus in blood ; x 1000 (Frankel and Pfeiffer). 

a wide capsule. The capsule, however, is best shown in 
cover-glass preparations from infected tissues (Figs. 88, 89). 
Gram- negative; not motile. 

Blood-serum. — After twenty-four to thirty-six hours in 
the incubator the colonies appear as translucent, colorless, 
rounded, convex elevations, resembling drops of mucus. If 
few in number, they may attain a diameter of 2-3 mm. 
They are viscid, and when touched with the platinum wire 
may be drawn out into threads. The water of condensation 
may become thick or viscid from the growth of the organism 
in it. 



332 PATHOLOGICAL TECHNIQUE. 

Glucose Agar-agar Stab. — Growth along the line of inocu- 
lation, with the production of a few gas-bubbles in the medium. 
Bouillon. — Clouded with the formation of a thin pellicle. 
Potato. — Thin, colorless, viscid layer. 
Litmus-milk. — Turned red and coagulated. 
Gelatin. — Growth not remarkable. 

There apparently exists a number of varieties of aerobic capsu- 
lated bacilli differing from one another only in non-essential par- 
ticulars. The organism here described is to be taken as a type of 
a group of closely-related bacteria of which the bacillus pneu- 
monic? of Friedldnder is a well-known member. 




Fig. 89. — Bacillus mucosus capsulatus; cover-glass preparation from sputum. 
Stained by W. H. Smith's method; X 1500 (W. H. Smith; photo, by L. S. 
Brown"). 



Pathogenesis. — White mice, rabbits, and guinea-pigs die 
from septicemia in a short time after inoculation, the capsule 
bacilli being present in the organs and blood of the heart in 
large numbers. 

White mice die in twenty-four hours to three days. Rab- 
bits inoculated in the ear vein and guinea-pigs inoculated in 
the peritoneal cavity may die within twenty-four hours. 

Subcutaneous inoculation of the animals last named leads 
only to local suppuration. The lesions produced by this 
organism consist in marked congestion of the superficial 
veins, hemorrhage into the lymphatic glands, and enlarge- 
ment and softening of the spleen. In the guinea-pig a 
hemorrhagic condition of the suprarenal capsules is present. 



PATHOGENIC BACTERIA AND FUNGI. 



333 



and in the peritoneal cavity there may be a small amount 
of clear, rather viscid fluid containing the bacilli in large 
numbers. 

The organs on microscopic examination may show pecu- 
liar areas in which the cells and nuclei are shrunken and in 
which the bacilli are aggregated. 

Occurrence. — This organism or closely related forms may 
be met with in broncho- or lobular pneumonia and in inflam- 
matory conditions of the air-passages generally. It may 




Fig. 90. — Bacillus mucosus- capsulatus, from a culture ; x 1000 (Wright and 

Brown). 

also be present in the upper air-passages of healthy individ- 
uals. It has been observed in inflammations of the middle 
ear, in empyema, meningitis, endocarditis, peritonitis, and in 
pus formations. In fatal infections the blood-stream may be 
found invaded by the organism. It is held by some bacteri- 
ologists that the members of this group may be the infective 
agents in genuine croupous pneumonia in rare instances. 
Representatives of this group have been found in the soil, 
in the air, and in contaminated water. The following 
method is recommended for staining the capsules in sections : 



334 PATHOLOGICAL TECHNIQUE. 

i. Stain for twenty-four hours in the incubator in the fol- 
lowing solution : 

Concentrated alcoholic solution of methyl-violet, 50 ; 
Distilled water, 100; 

Glacial acetic acid, 10. 

2. Wash out in a 1 per cent, solution of acetic acid. 

3. Alcohol. 

4. Oil. 

5. Xylol balsam. 

If the process of decolorization is stopped at the right 
moment, the capsules will be pale blue, while the bacilli 
will be stained deep blue. 

Bacillus of Rhinoscleroma. — Method of staining cap- 
sules in sections of tissues hardened in alcohol (Wolko- 
witsch) : I. Stain twenty-four to forty-eight hours in aniline- 
methyl-violet. 

2. Wash off in water. 

3. Iodin solution one to four minutes. 

4. Absolute alcohol. 

5. Oil of cloves, which removes still more of the color. 

6. Xylol. 

7. Xylol balsam. 

According to Wolkowitsch, the hyaline masses in rhino- 
scleroma stain intensely with methyl-violet, gentian-violet, 
methylene-blue, and fuchsin; less with safranin, and not at 
all with hematoxylin. Eosin stains them well. Double 
staining with hematoxylin and eosin is therefore to be 
recommended highly. 

Bacillus Aerogenes Capsulatus. — Synonyms: Ba- 
cillus Welchii; Bacillus perfringens. Grows best under 
anaerobic conditions. 

Morphology. — Bacilli of about the thickness of the anthrax 
bacillus, variable in length, but usually 3 to 6 /x long. Ends 
rounded or square cut. Occurs singly, in 'pairs, in clumps, 
and sometimes in short chains, less frequently in threads 
and long chains. 

May show unstained spots or deeply staining granules in 
the protoplasm. Capsules may be frequently demonstrated 
in the specimens from the tissues, and sometimes in agar- 
agar cultures. Gram-positive; not motile. 



PATHOGENIC BACTERIA AND FUNGI. 335 

Forms spores situated near one end when other bacteria 
are growing with it, but rarely does so in pure cultures. 

Colonies in anaerobic cultures are grayish to brownish- 
white, with a central darker spot by transmitted light. In 
time they may attain a diameter of 2 to 3 mm. or more. 
Colonies in the depths are spherical or oval, sometimes pre- 
senting knob-like or feathery projections. 

Effects on Animal Tissues. — Not pathogenic for rabbits. 

If a rabbit that has received 0.5 to 1 c.c. of a bouillon 
culture injected into the ear-vein be killed immediately after- 
ward and the body kept for twenty-four hours at a tempera- 
ture of 1 8° to 20 C, or for four to six hours at a tempera- 
ture of 30 to 35 C, the vessels and organs will be found 
to contain a great quantity of gas and large numbers of 
the bacilli. The organism multiplies post-mortem in the 
blood of the animal and produces the gas. This effect 
upon the tissues of the dead animal is characteristic of the 
bacillus. 

The subcutaneous inoculation of guinea-pigs with young 
cultures may produce fatal gas phlegmons. The hemor- 
rhagic fluid from the dead animal is 
virulent for other guinea-pigs, and may 
be virulent for rabbits. 

Gas-production is marked in agar-agar 
and gelatin cultures containing glucose. 
The gas produced burns with a blue 
flame and is odorless. 

Gelatin is liquefied slowly and to a 
limited extent. ^ ^^ZfZ' 

Glucose Bouillon. — Diffusely clouded glass pre paration from the 
at first, later becoming clearer, with an spleen, stained by w. 
abundant whitish, more or less viscid H ' Sm ^' s ™ eth ° d; . * 

' 1500 (W. H. Smith; 

sediment. photo, by L. S. Brown). 

Milk. — Coagulated, the clot being 
firm, retracted, and furrowed with the marks of gas- 
bubbles. 

Potato. — Growth thin, moist, and grayish-white, or it may 
not be visible. 




33^ 



PATH OLD GICA L TE CHNIQ UE. 



The vitality of the organism depends upon the character 
of the culture-medium and the mode of cultivation. It sur- 
vives longer when cultivated by Buchner's method (see 
page 223) than when cultivated under hydrogen. Cultures 
on glucose media are shorter lived than those on plain media. 

Occurrence. — Occurs at autopsies in which gas-bubbles are 
present in the larger vessels, accompanied by the formation 
of numerous small cavities in the liver containing gas. It 
has been found also in shell wounds, emphysematous 
phlegmons, in puerperal sepsis, in peritonitis, and in other 
conditions. It is a normal inhabitant of cultivated soil and 
of feces. 

Bacillus of Tetanus.— This bacillus will not grow in 
the presence of oxygen. 




Fig. 92. — Tetanus bacilli showing flagella, from a preparation stained by Dr. 
Hugh Williams; X 2000 (Wright and Brown). 

Morphology. — Slender rods with rounded ends, which may 
grow into long threads. In the incubator spores are rapidly 
formed. These are round, wider than the bacillus, and are 
situated at the end of the rod, giving the appearance of a 
drum-stick or a round-headed pin (Fig. 96). Gram-positive; 
motile. 

The colonies in anaerobic glucose-gelatin cultures appear 
after several days as small clumps of interlacing fibrillar from 
which delicate filaments radiate into the gelatin, which is 
slowly liquefied. 



PATHOGENIC BACTERIA AND FUNGI. 



337 



The colonies in simple anaerobic glucose-agar plate cul- 
tures (see page 223) appear after twenty-four to forty-eight 
hours in the incubator, as groups and masses of long fila- 
ments radiating from a center (Fig. 92). 

Glucose-gelatin Stab. — Growth along the line of inocula- 
tion, beginning 2 or 3 cm. below the surface, with delicate 
filaments radiating laterally into the gelatin (Fig. 95). Liq- 
uefaction and gas-production occur. 

In deep-stab cultures in glucose-agar faintly alkaline to 
litmus (see Fig. 94) growth appears first all along the line of 
inoculation to within about 1 cm. of the surface after about 
twenty-four hours in the incubator. Later, lateral out- 
growths extend into the medium from all along the line of 




Fig. 93. — Colony of tetanus bacilli in anaerobic glucose-agar plate ; low mag- 
nifying power (Wright and Brown). 



inoculation below a point about 1 cm. below the surface. 
In the portion of the line of inoculation above this, growth 
is frequently observed up to the surface, but without lateral 
outgrowths. The growth eventually assumes the appear- 
ance of an inverted pine tree. A peculiar feature of the 
culture is the appearance of a brown pigmentation in the 
culture-medium in its upper layers in the form of a flat or 
cone-shaped zone. A small quantity of gas may be pro- 
duced. 

If the agar has a reaction of about 1 per cent, normal 

acidity to phenolphthalein (see p. 203) growth appears 
22 



338 



PA THOL O GICA L TE CHNIQ UE. 



along the line of inoculation and spreads through the me- 
dium as a cloudiness extending to within a few millimeters 
of the sursace. The employment of glucose culture-media 
not older than a week or so seems to be important for 
success in cultivating this organism. 

In the vegetative form the organism is sluggishly motile. 
It has numerous flagella. It is stained by Gram's method. 




u 







Fig. 94. — Tetanus bacillus. Stab- Fig. 95.— Bacillus of tetanus : six- 
culture in glucose-agar. In the upper days-old stab-culture in glucose-gelatin 
layers of the medium the peculiar (Frankel and Pfeiffer). 
brownish coloration is shown. 

Glucose-bouillon. — Growth appears first, after twenty-four 
to forty-eight hours, as a diffuse cloudiness. Later the fluid 
becomes clear, and a grayish sediment collects at the bottom 
of the tube. Only a small amount of gas is produced. 

Pathogenesis. — Subcutaneous inoculation of mice at the 
root of the tail gives rise to tetanic symptoms in twenty- 
four hours, followed by death in two or three days. 



PATHOGENIC BACTERIA AND FUNGI 339 

Guinea-pigs and rabbits are also susceptible to the infec- 
tion, the period of incubation in these animals being twenty- 
four to thirty hours in the former and two to three days in 
the latter animal, after subcutaneous inoculation. The symp- 
toms of tetanus appear first in the extremities nearest the 
point of inoculation. In mice the hind legs become rigidly 
extended backward. At the autopsy the bacillus is to be 
found only at the point of inoculation, and may be difficult 
or impossible to demonstrate there. 

Occurrence. — Found in the soil, and often in the feces of 
herbivorous animals. In cases of tetanus the bacillus is to 
be found only in the wound or at the point of inoculation. 
It does not invade the blood-current. 

The bacillus of tetanus acts by the production of a " toxin " 
or " toxalbumin!' This is also produced in cultures. It may 
be demonstrated in the bacteria-free filtrate of bouillon cult- 
ures some days or weeks old. A very few drops of this 
fluid will give rise to fatal tetanus in a mouse. 

Method of Isolation. — Tetanus bacilli will grow in aero- 
bic culture if other bacteria are growing with them. Since 
tetanus wounds usually contain other bacteria, all that is 
necessary to obtain an impure culture of the tetanus bacillus 
is to inoculate an ordinary blood-serum culture-tube (see 
page 198) with material from the wound. After several 
days or a week in the incubator, if tetanus bacilli are present 
they can be recognized by cover-glass preparations from 
the growth in the tube by their morphology and spore- 
formation (see Fig. 96). There will also be a peculiar, 
stinking odor about the culture. The isolation of the 
tetanus bacillus is now to be proceeded with as follows : 
Mix a loopful of the mixed growth on blood-serum with 
a tube of sterile bouillon, and heat in a water-bath for at 
least fifteen minutes at 8o° C, then make anaerobic cultures 
from this (see Anaerobic Methods, page 220), taking several 
loopfuls for inoculation. 

If other spore-bearing bacilli are present in the mixed 
culture in the blood-serum tube, it will be necessary to use 
some form of anaerobic culture on a solid medium in order 



340 



PA THOL GICA L TE CHNIQ UE 



to obtain separate colonies of the tetanus bacillus for further 
cultures. 

The bacillus may be isolated from wounds and from the 
soil by inoculation of mice subcutaneously, and proceeding 
as above described with material from the seat of inoculation. 




Fig. 96. — Spore-bearing tetanus bacilli in an impure culture on blood-serum 
from a case of tetanus. In the bacillus on the extreme left the beginning of 
spore-formation is shown (Wright and Brown). 

Bacillus of Malignant Edema. — This bacillus will 
not grow in the presence of oxygen. 




Fig. 97. — Bacillus of malignant edema from the edema fluid of a guinea-pig 
inoculated with garden-earth ; X 1000 (Frankel and Pfeiffer). 

Morphology. — Rather large bacilli, sometimes growing into 
threads (Fig. 97), but occurring frequently in pairs, in which 



PATHOGENIC BACTERIA AND FUNGI. 



341 



the proximal ends are square, while the distal ends are 
rounded. Forms oval spores in the middle of the rod, 
which may give the rod a spindle or oval shape. Gram- 
negative; motile. 

The colonies in anaerobic glucose-gelatin cultures appear 
as spheres of cloudy liquefied gelatin marked by delicate 
radiating streaks. Gas-bubbles are formed in the medium 
(Fig. 98). 




Fig. 98. — Bacillus of malignant edema; colonfes growing in glucose-7elatin (Frankel 

and Pfeiffer). 

In glucose-agar the colonies appear as hazy points made 
up of interlacing filaments and resembling very much the 
colonies of the tetanus bacillus. 

Pathogenesis. — Subcutaneous inoculation of mice, guinea- 
pigs, and rabbits is followed by death in from sixteen to 
forty-eight hours, depending upon the animal, mice being 
most susceptible. The typical lesions are extensive sub- 
cutaneous edema containing gas-bubbles and more or less 



1 



342 PATHOLOGICAL TECHNIQUE. 

blood, and enlargement of the spleen. The bacilli are found 
in the edema, in the viscera, and on the serous surfaces of 
the organs, but not in the blood of the heart if the exam- 
ination be made immediately after death, except sometimes 
in mice. The organism is not capable of multiplying in the 
living blood, owing to the presence of oxygen. In inoculat- 
ing subcutaneously a deep pocket should be made in the 
skin, and the material for inoculation introduced into the 
tissue as far away from the opening as possible. This is to 
prevent the access of too much oxygen to the organism. 

Slightly motile. Flagella may be demonstrated by special 
staining methods. 

The bacilli in tissues are stained by Gram's method, but 
in cultures most of them are decolorized by it, probably 
because of rapid degenerative changes in them. 

Growth in anaerobic agar- agar and bouillon culture is 
good, but not characteristic. 

Occurrence. — Widely distributed in the soil and in putre- 
fying substances. Only a very few cases are on record of 
infection in man by this bacillus. 

Bacillus Tuberculosis. — Synonyms: Tubercle bacillus ; 
Bacillus of Koch. 

Morphology. — Slender rods, usually shorter than when 
observed in sputum, and in fresh cultures staining homo- 
geneously; in older cultures presenting a segmented or 
irregularly stained appearance. They frequently occur in 
pairs of short rods and in closely adhering clumps and 
strands. When once stained with fuchsin or gentian-violet 
they are not decolorized by treatment with Gabbet's solution 
or with a 20 per cent, solution of any of the mineral acids, 
followed by alcohol. In the sputum of pulmonary tuber- 
culosis the bacillus sometimes occurs in filaments which 
branch. On this account the organism is considered by 
many to belong to the group of the streptothrices. 

Stained by Gram's method. Not motile. Does not form 
spores. 

Blood-serum. — After three or four weeks in the incubator 
the colonies appear as dry, cream-colored, granular, slightly 
elevated patches with irregular margins, 1 to 2 mm. in diam- 



PATHOGENIC BACTERIA AND FUNGI. 



343 



eter. They may become confluent, to form a dense, dry, 
granular mass with irregular surface and of a creamy-white 
color. The growth is very friable, but coherent, and may 
be picked up in clumps on the platinum wire. The first 
generation from tissues is very slow in developing, but suc- 
ceeding generations grow more rapidly, and may form a 
wrinkled, dry, cream-colored membranous layer on the sur- 
face of the medium. 

Glycerin Agar-agar Slant. — Growth similar to that on 
blood-serum, but not so vigorous. By continued inocu- 
lation of this medium through a number of generations, 




Fig. 99. — Branched tubercle bacilli from sputum; X 2 °°° (Wright and Brown). 

however, the organism may eventually grow luxuriantly 
upon it. 

Glycerin Bouillon. — Growth on the surface in the form of 
floating patches or as a membrane similar in appearance 
to the growth on blood-serum. The growth sinks to the 
bottom from time to time. The glycerin-bouillon culture is 
best contained in Erlenmeyer flasks, filled to such a depth 
as to give a wide surface to the fluid and thus permit the 
access of plenty of oxygen to the growth. 

Potato. — The growth is not remarkable. 

Agar-agar or bouillon not containing glycerin is not suit- 
able for the cultivation of this bacillus. 

Pathogenesis. — The inoculation of guinea-pigs or rabbits 
by any method is followed by the development of general 
miliary tuberculosis. Guinea-pigs are most susceptible. 
These animals usually survive about two or three months, 
with marked emaciation. The lesions in the spleen and liver 



344 PATHOLOGICAL TECHNIQUE. 

in the guinea-pig are characterized by extensive areas of 
necrosis not confined to the tubercular tissue, large parts 
of these organs being transformed into a firm yellow, opaque, 
friable material. 

Isolation of the Bacillus Tuberculosis from Tubercular 
Lesions. — The tubercular lesions in human tissues are not 
ordinarily favorable for the isolation of the bacillus, on 
account of the frequent presence of other bacteria in them 
and because of the small number of tubercle bacilli usually 
present in tissue otherwise suitable. The best method of 
procedure is to inoculate a guinea-pig subcutaneously in the 
abdominal wall with tubercular material, and after four to 
six weeks, when the inguinal lymphatic glands have become 
enlarged, to kill the animal and make cultures on suitable 
media from tuberculous lymphatic glands. The object of 
killing the animal, rather than allowing it to die sponta- 
neously, is to secure fresh tissue and to avoid the chance 
of an invasion of the lesions by other bacteria. 

For the cultivation of tubercle bacilli from tubercular 
lesions, M. Dorset highly recommends his egg-medium, 
which is prepared as follows : 

Fresh eggs are broken under aseptic precautions into a 
wide-mouthed sterile flask, and the white and yolk 'mixed 
thoroughly. To every four eggs add 25 c.c. of sterile water. 
Any foam may be removed by straining the mixture through 
a sterile cloth. The mixture is then run into sterile tubes, — 
about 10 c.c. into each tube, — and slowly hardened in the 
form of " slants " in a blood-serum oven at a temperature 
of 73 to y6° C. This degree of temperature should be 
maintained for four or five hours on three successive days. 
On the first two days the temperature is maintained at 73 ° 
C, and on the third day at 76 C. Just before inoculating 
the medium three or four drops of sterile distilled water 
should be added to each tube to supply the moisture 
required for the satisfactory development of the tubercle 
bacillus. 

After inoculation the tube should be placed in the incu- 
bator at 38 C. in an inclined position, so that the surface of 



PATHOGENIC BACTERIA AND FUNGI. 345 

the medium may keep moist. Colonies first become visi- 
ble after seven or eight days in the incubator. 

A number of tubes are to be inoculated, say three or four, 
from each of the two or three glands, a large quantity of 
material being spread upon the surface of each tube. Great 
care is to be exercised to avoid contamination with other 
bacteria in preparing these cultures. The culture-tubes used 
should contain freshly prepared moist medium, and imme- 
diately after inoculation should be sealed air-tight to prevent 
evaporation. This may conveniently be done by first cutting 
off the projecting portion of the cotton stopper and insert- 
ing a cork into the mouth of the tube in such a way as to 
push the cotton stopper before it. 

In order to prevent the invasion of fungi from the cotton, 
the neck of the tube should be heated in the Bunsen flame 
until the cotton begins to brown before inserting the cork, 
which should also be charred in the Bunsen flame before in- 
sertion. The tubes may also be sealed with wax or paraffin 
or covered with small rubber caps. 

Wolbach and Ernst have noted the following chief points 
of difference between tubercle bacilli from human and from 
bovine lesions when cultivated on Dorset's egg medium. 
The human cultures as compared with the bovine cultures 
grow more profusely and the membranous layer that is 
formed is more nodular, drier looking, less translucent, more 
adherent to the medium, harder, and more difficult to break 
up with the platinum wire. The human bacilli generally 
show a slightly greater variation in length and thickness 
than do the bovine bacilli. 

S. A. Petroff's Method for the Cultivation of Tubercle 
Bacilli from Sputum and Feces. — Equal parts of sputum and 
3 per cent, sodium hydrate solution are mixed and incu- 
bated for twenty to thirty minutes to liquefy the sputum. 
The alkali is then neutralized with normal hydrochloric acid, 
the mixture centrifuged, and cultures sown on a special 
medium which is prepared as follows: 

An infusion of beef or veal is prepared by mixing 500 grams 
of finely ground meat with 500 c.c. of 15 per cent, glycerin 
solution, and after twenty- four hours' standing in the ice- 



346 PATHOLOGICAL TECHNIQUE. 

chest the mixture is passed through a sterile meat press and 
the fluid collected in a sterile beaker. This infusion is 
mixed with an equal volume of whole eggs and sufficient of 
a i per cent, alcoholic solution of gentian violet to make a 
dilution of I : 10,000. The mixture is tubed and inspissated 
in the form of "slants" at 85 C. and then heated for not 
more than one hour at 75 C. on each of the two following 
days. 

The shells of the eggs are sterilized by immersing in 
70 per cent, alcohol for ten minutes or by pouring hot water 
over them. 

Before admixture with the infusion the whites and yolks 
of the eggs are beaten up in a sterile beaker and filtered 
through sterile gauze. 

Feces are. mixed with three volumes of water and filtered 
through gauze. The filtrate is then saturated with sodium 
chloride, allowed to stand for a half-hour, when the film 
at the surface containing the bacteria is transferred to a 
bottle, shaken up with equal parts of normal sodium hydrate 
solution, and left in the incubator for three hours, shaking 
up every half-hour. The mixture is then neutralized to 
litmus-paper with normal hydrochloric acid, centrifuged, 
and cultures sown on the surface of the special medium. 

The Antiformin Method for Obtaining Pure Cultures of the 
Tubercle Bacillus. — Antiformin is the patented name for a 
solution consisting of equal parts of liquor sodae chlorinatae 
and a 15 per cent, solution of caustic soda. It quickly dis- 
solves mucus and the cells and fibers of animal tissues, and 
also has the remarkable property of destroying all bacteria 
except tubercle bacilli and other acid-fast bacilli. 

Lawrason Brown and Daniel Smith have used the follow- 
ing procedure with great success in cultivating tubercle 
bacilli from the sputum in a series of cases : 

Equal parts of a 30 per cent, aqueous solution of anti- 
formin and sputum are thoroughly mixed in a sterile centri- 
fuge tube and allowed to stand at room-temperature for one 
hour. The tube is then centrifugalized, the supernatant fluid 
decanted, and the sediment mixed with sterilized distilled 
water. This is again centrifugalized, and the whole process 



PATHOGENIC BACTERIA AND FUNGI. 347 

is carried out three times. The sediment is then streaked 
over the surface of Dorset's egg medium and placed in the 
incubator. 

Cultures from tubercular tissue may also be obtained by a 
similar procedure, the tissue being ground up in a mortar 
with a 15 or 20 per cent, solution of antiformin, or frozen 
sections made of it and placed in the same solution. When 
the tissue has been dissolved, which occurs in the course of 
a few minutes, the solution is to be centrifugalized, the sedi- 
ment washed, and cultures made from it as above described 
for sputum. 

A. S. Griffith has found that it is not necessary to wash 
the bacilli free from antiformin in order to obtain cultures. 
Antiformin is mixed with the sputum in the proportion of 
15 per cent, and a loopful of the mixture is spread over the 
surface of the egg medium in tubes, three tubes being sown 
at intervals of one or two minutes. Thereafter tubes are 
sown at intervals of five minutes if the sputum remains 
undissolved and mucinous. After twenty minutes or after 
complete solution the tubercle bacilli will not be viable. 

Occurrence. — In tubercular lesions generally and in the 
sputum of pulmonary phthisis, in the urine in many cases 
of genito-urinary tuberculosis, and in the feces in intestinal 
tuberculosis. The tuberculosis of cattle and of birds is due 
to different varieties of this bacillus. 

Does not multiply outside of the body except in cultures. 

May occur on the surface of objects contaminated with 
the excreta of tuberculous individuals or in the dust of places 
inhabited by such individuals. 

Diagnosis. — For clinical purposes the tubercle bacillus 
may be identified in cover-glass preparations by means of 
the special methods of staining, which depend upon the fact 
that the bacillus tuberculosis, when once thoroughly stained 
with an aniline dye, does not give up its stain in the presence 
of acids, as nearly all other bacteria do. The bacillus tuber- 
culosis may therefore be identified even among a mixture of 
other bacteria by this property, taken in connection with its 
morphology, in most of the routine work of the pathological 
laboratory. Practically, the only other bacilli with which it 



34^ PATHOLOGICAL TECHNIQUE. 

may be confounded are the bacillus of leprosy and the 
smegma bacillus, both of which, when stained, resist the 
decolorizing action of acid. It may be differentiated from 
the smegma bacillus by the fact that it is not decolorized by 
alcohol (95 per cent.) after the usual treatment with acid, 
while the smegma bacillus is decolorized under these circum- 
stances. 

As a rule, the differential test with alcohol need only be 
applied in the examination of urine and the material derived 
from about the external genitalia, especially in the case of 
females. 

The differentiation from the bacillus of leprosy by certain 
quantitative differences in staining reactions has been at- 
tempted, but it is very unsatisfactory, and it is doubtful if 
there is as yet any reliable method of distinguishing between 
these two organisms, considered by themselves. 

Examination of Sputum for Tubercle Bacilli.— 
The morning sputum should be taken for examination. 
Select one of the dense, grayish- white particles, and with 
the aid of small- pointed forceps or the platinum wire rub it 
over the surface of a cover- glass, breaking it up as much as 
possible. The material should be spread in a very thin layer. 
The preparation is next to be " fixed " in the ordinary way 
described for cover-glass preparations (see p. 234), and is 
then to be treated as follows : 

1. Stain in carbol-fuchsin solution, steaming for one minute 
over the Bunsen flame, with the staining solution thoroughly 
covering all the surface of the cover-glass. None of the 
surface of the cover-glass should be allowed to become dry 
by evaporation, as this causes a precipitate to form, but more 
of the staining fluid should be added from time to time to 
keep it completely covered as evaporation occurs. The 
object of the heating is thoroughly to impregnate the bacilli 
with the dye. 

2. Wash in water. 

3. Decolorize in a 30 per cent, aqueous solution of con- 
centrated nitric acid until the red color disappears. Do not 
allow the acid to act on the preparation longer than a few 



PATHOGENIC BACTERIA AND FUNGI. 349 

seconds. The solution should also be applied to the un- 
charged side of the cover-glass to remove any dried stain 
which may have collected thereon. 

4. Wash thoroughly in water. 

5. Wash in 95 per cent, alcohol for thirty seconds. 

6. Wash in water. 

7. Stain in Loffler's methylene-blue solution for thirty 
seconds. 

8. Wash in water, dry, and mount in xylol balsam. 

The tubercle bacilli are stained red and the nuclei of cells 
and other bacteria are stained blue. 









~N, 



Fig. 100. — Tubercle bacilli in sputum (carbol-fuchsin and methylene-blue) 

(Vierordt). 

Antiformin Method. — The finding of tubercle bacilli in sputum 
is greatly facilitated by making cover-glass preparations, as above 
described, from the sediment obtained by the antiformin method 
described on page 346. 

In order to destroy any extraneous tubercle bacilli or other acid- 
fast bacilli which may be in the centrifuge tube, just before use 
the interior of the tube is to be thoroughly exposed to the action 
of concentrated sulphuric acid saturated with potassium bichro- 



350 PATHOLOGICAL TECHNIQUE. 

mate, after which the tube is to be thoroughly washed with dis- 
tilled water. Sputum cups, or other receptacles of crockery or 
of glass used to collect the sputum, should be treated in the same 
way. 

It is of the greatest importance to be sure that the distilled 
water used does not contain acid-fast bacilli which sometimes de- 
velop in it. 

As antiformin does not kill tubercle bacilli, the centrifuge tube 
should be sterilized after use. 

In a very few cases of gangrene of the lung bacilli like smegma 
bacilli have been found in the sputum. These may be mistaken 
for tubercle bacilli {vide ante). 

Tubercle Bacilli in Urine. — The sediment of the urine 
should be examined. This may be rapidly thrown down by 
the centrifuge. If it is abundant, the urine should be first 
centrifuged at low speed, and then transferred to another 
tube and centrifuged for an hour at high speed. It is 
important that the tubes be mechanically clean and free 
from any bacilli from previous specimens of urine. With 
the sediment cover-glass preparations are to be made and 
stained as described for sputum. Especial care should be 
taken to wash thoroughly in alcohol after the decolorization 
with acid, in order to decolorize any smegma bacilli that 
may be present. (See remarks on Diagnosis, page 347.) 
Because smegma bacilli may be mistaken for tubercle bacilli 
and because the tubercle bacilli may be so few as to escape 
observation, the inoculation of a guinea-pig with the sedi- 
ment is the better test for the presence of tubercle bacilli 
in the urine. If, however, the urine be obtained by cath- 
eter from a ureter, the first objection is practically elimi- 
nated. 

For inoculation the urine should be collected in sterilized 
vessels and immediately centrifugalized in sterilized tubes. 
The sediment is then to be injected subcutaneously into a 
guinea-pig with a sterilized syringe. (See page 229.) In 
many cases centrifugalizing is not necessary. 

Tubercle Bacilli in Tissues, Pus, and Feces.— The bacilli 
may be demonstrated in the following ways : 

1. By the staining of the bacilli in sections of tissue by the 
special methods described on pages 351 to 353 Frozen sec- 



PATHOGENIC BACTERIA AND FUNGI. 35 I 

tions prepared by the method elsewhere described may be 
employed. 

2. By making cover-glass preparations and staining as de- 
scribed for sputum. These preparations may be made directly 
from the material ; but if the bacilli are few, as is usually the 
case, they should be made from the sediment obtained by the 
antiformin method described on page 346. This method is 
also applicable to fixed and hardened tissue, even if it has 
been imbedded in paraffin. The paraffin should be thor- 
oughly removed from the sections by means of xylol, fol- 
lowed by absolute alcohol, before placing them in the solu- 
tion of antiformin. 

The precautions against error from the presence of ex- 
traneous tubercle bacilli, or other acid -fast bacilli, are to be 
taken which are described in connection with the application 
of this method to the examination of the sputum. 

3. By the inoculation of guinea-pigs with the material or 
sediment obtained by the antiformin method. The inocula- 
tion is best made subcutaneously in the abdominal wall, 
either with a hypodermic syringe, if the material be fluid, or 
if it is in the form of tissue, by inserting a small piece be- 
neath the skin. Material obtained on a swab may also be 
used for inoculation by introducing the infected swab beneath 
the skin and moving it back and forth a few times. If tuber- 
cle bacilli are present in the material, the animal will show 
enlargement of the inguinal lymphatic glands in about three 
weeks, and will usually die of miliary tuberculosis in the 
course of six to ten weeks. If necessary, the glands in the 
inguinal region may be examined histologically after three 
weeks for the presence of tubercular lesions, or examined 
by cover-glass preparations for tubercle bacilli. 

Bacteria that Stain by the Tubercle Bacillus Method. 

Tubercle bacillus ; 
Leprosy bacillus ; 
Smegma bacillus. 

The important point about staining tubercle bacilli is to 
stain them deeply enough in the beginning ; then there is 



352 PATHOLOGICAL TECHNIQUE. 

little danger of their fading in the subsequent steps of con- 
trast staining. It is probable that carbol-fuchsin, used hot, 
is the most powerful stain we have for this purpose. If the 
solution is steamed, generally on the slide, one to five min- 
utes are probably sufficient for all purposes. Tubercle bacilli 
stain well, not only after alcohol, but also after most of the 
other fixing reagents, such as corrosive sublimate, Zenker's 
fluid, Flemming's solution, etc. 

Ehrlich's Method. — i. Stain paraffin sections in aniline- 
fuchsin or methyl-violet for half an hour to twenty-four 
hours, or for one to five minutes if solution is heated to 
steaming. 

2. Wash in water. 

3. Decolorize in 20 per cent, nitric acid one-half to one 
minute. 

4. Wash in 70 per cent, alcohol until no more color is 
given off. 

5. Contrast-stain in a saturated aqueous solution of methy- 
lene-blue or of Bismarck brown one to two minutes. 

6. Wash in water. 

7. Dehydrate in absolute alcohol. 

8. Xylol, xylol balsam. 

Ziehl-Neelson-Gabbet Method.— 1. Stain paraffin sec- 
tions in carbol-fuchsin solution, warming the solution so that 
it steams one to three minutes. 

2. Wash in water. 

3. Decolorize and stain for contrast in sulphuric-acid- 
methylene-blue solution one minute (see page 74). 

4. Wash in water. 

5. Absolute alcohol 

6. Xylol. 

7. Xylol balsam. 

This method is not suited to celloidin sections, because 
the celloidin retains too deep a blue stain. 

Kiihne's Method. — 1. Stain paraffin sections lightly in 
alum-hematoxylin. 

2. Wash in water. 



PATHOGENIC BACTERIA AND FUNGI. 353 

3. Stain in carbol-fuchsin one to five minutes if warmed; 
longer if cold. 

4. Wash in water. 

5. Aniline hydrochlorate, 2 per cent, aqueous solution, 
fifteen seconds. 

6. Wash in water. 

7. Absolute alcohol. 

8. Xylol. 

9. Xylol balsam. 

To Stain Tubercle Bacilli in Celloidin Sections. — L. 
Stain rather lightly in alum-hematoxylin. 

2. Wash in water. 

3. Dehydrate in 95 per cent, alcohol. 

4. Attach sections to slide by the ether-vapor method. 

5. Carbol-fuchsin two to five minutes steaming. 

6. Water. 

7. Orth's discharging fluid (acid alcohol) one-half to one 
minute. 

8. Wash thoroughly in several changes of water to re- 
move acid completely and to bring back blue color to nuclei. 

9. Alcohol 95 per cent, until fuchsin is entirely discharged. 

10. Aniline followed by xylol; or blot and treat with 
xylol. 

11. Xylol balsam. 

The advantages of this method are — that the celloidin is 
colorless ; the nuclei are stained blue ; the rest of the tissue 
is colorless; the tubercle bacilli stand out in sharp contrast. 
It is sometimes an advantage to bring out the cell-proto- 
plasm and the intercellular substance by staining the sec- 
tions, after decolorization in alcohol, in an aqueous solution 
of orange G or methyl-orange for a few seconds. 

Bacillus Of I^eprosy. — This bacillus resembles closely 
the tubercle bacillus in morphology and staining reactions. 
It is somewhat less resistant than that bacillus to decoloriza- 
tion by acids. 

Cultures. — There is no satisfactory evidence that this or- 
ganism has ever been obtained in pure culture. 

23 



354 PATHOLOGICAL TECHNIQUE. 

Pathogenesis. — The Japanese waltzing mouse is the only 
animal known to be very susceptible to inoculation with this 
bacillus. The inoculation of this animal is followed by ex- 
tensive and widespread nodular lesions containing large 
numbers of bacilli, and very similar in histological character 
to those of the disease in man. 

Occurrence. — The bacillus often grows in enormous num- 
bers in the lesions, and chiefly in the cytoplasm of mono- 
nuclear cells, where they often lie parallel to one another in 
bundles. They may also be found in nerves and in nerve- 
cells. In lesions of the nasal mucosa they may be demon- 
strated in the secretion. 

To Stain the Bacillus of Leprosy in Sections. — The bacillus 
of leprosy stains more easily than the tubercle bacillus. 
Simple aqueous solutions of the aniline dyes are sufficient. 
The Gram-Weigert stain gives a brilliant picture. The 
same methods can be employed as for tubercle bacilli if a 
differential stain is desired. A method recommended by 
Elexner will be found very useful. 

1. Stain in alum-hematoxylin so as to get a sharp nuclear 
stain. 

2. Wash in water. 

3. Carbol-fuchsin two to five minutes steaming, or thirty 
to sixty minutes cold. 

4. Water. 

5. Treat on the slide with iodin solution one-half to one 
minute. 

6. Water. 

7. Blot; clear and differentiate in aniline oil. 

8. Xylol; balsam. 

Baumgarten gives the following . differential stain for 
leprosy bacilli : 

1. Stain six to seven minutes in a dilute solution of fuch- 
sin (5 drops of a concentrated alcoholic solution to a watch- 
glass of water). 

2. Discharge one-quarter minute in nitric acid alcohol 
(nitric acid I, alcohol 10).' 

3. Wash in water. 



PATHOGENIC BACTERIA AND FUNGI. 



355 



4. Contrast-stain in a saturated aqueous solution of meth- 
ylene-blue. 

5. Alcohol. 

6. Xylol. 

7. Balsam. 











*» 



Fig. ioi. — Bacillus of leprosy : section through a cutaneous nodule, showing the 
bacilli in the tissue; X 750 (Wright and Brown). 

While leprosy bacilli stain readily by this method, tubercle 
bacilli will not stain in so short a time. 
Spirillum of Asiatic Cholera (Comma Bacillus).— 

Morphology (Figs. 102, 103). — In fresh cultures the organism 
appears usually as a slightly curved rod somewhat shorter 
than the tubercle bacillus, but much thicker. The curving 
of the rod varies, being very marked in some individuals 
and absent in others. Sometimes two rods are joined end 
to end with their convexity pointing in opposite directions, 
or moderately long, undulating threads may be found. It 
seems probable that the curved rods represent the segments 
of a spirillum, and hence the name of the organism. 

In cultures some days old degenerated and atypical forms 



356 PATHOLOGICAL TECHNIQUE. 

are found (involution forms). The organism is motile, and 
a single flagellum is attached to the end of the rod. 



-4 *i$j*>*\ , z* 5 ^ 

1 r> r^\^ 



ft§ 



Jy-Z/ 



.Mi 



' .*! 









Fig. 102. — Spirillum of Asiatic cholera, from a bouillon culture three weeks old, 
showing long and degenerate forms; X iooo (Frankel and Pfeiffer). 




Fig. 103. — Spirillum of Asiatic cholera, showing the flagella ; X 1000 (Giinther). 

It is not stained by Gram's method; motile. 
Colonies on Gelatin Plates (Fig. 104). — After twenty-four 
to forty-eight hours at a temperature of 20 to 22 C. the 



PATHOGENIC BACTERIA AND FUNGI. 357 

largest colonies will appear as masses of indefinite granular 
material lying in circular areas of liquefied gelatin in which 
granular shreds are scattered. Within the next twenty-four 
hours the areas of liquefaction increase, and the colonies ap- 



: 






Fig. 104. — Developmental stages of colonies of the spirillum of Asiatic cholera 
at 20 to 22 C. on gelatin; X about 75 diameters (Abbott): a, after sixteen to 
eighteen hours; b, after twenty -four to twenty-six hours; c, after thirty-eight to 
forty hours; d, after forty-eight to fifty hours; e, after sixty-four to seventy hours. 

pear under the low power " as a dense granular mass sur- 
rounded by an area of liquefaction through which can be seen 
granular prolongations of the colony, usually extending ir- 
regularly between the periphery and the central mass " (Ab- 
bott), while the margin of the liquefied area is marked by 
delicate radiating filaments closely packed together. 

The colonies on agar-agar plates are not characteristic. 
Growth is rapid. 

Gelatin Stab. — Growth all along the line of inoculation 
with liquefaction at the surface in funnel form after forty- 
eight hours. The liquefaction proceeds in such a manner 
that the liquefied area has a smaller diameter at the surface 
than immediately beneath, and, owing to the fact that the 
liquefied gelatin does not fill the cavity, a space is left be- 
tween the surface of the medium and the surface of the lique- 
fied gelatin so that the appearance of an air-bubble is pro- 
duced. Along the deeper portions of the line of inoculation 
the liquefaction is slow. 

Bouillon. — Diffusely clouded. A thin pellicle forms on the 
surface after a time. 

Litmus-milk. — Turned red and coagulated. 



358 PATHOLOGICAL TECHNIQUE. 

Indol-production. — In cultures in Dunham's pepton solu- 
tion or in the pepton solution of Koch (2 per cent, pepton 
and I per cent, sodium chlorid) a rose-color is produced by 
the addition of sulphuric acid alone. (Concentrated c. p. 
acid is to be employed, as in the test for indol-production by 
the bacillus coli communis.) The production of the rose-color 
without the addition of the sodium nitrite shows that nitrites 
as well as indol are formed by the growth of the organism 
in the pepton solution. The reaction can be obtained in 
cultures which have been but eight hours in the incubator. 

Potato. — Thin, dry, grayish-white growth which does not 
spread over the surface. 

Pathogenesis. — The pathogenic effects of the cholera 
spirillum are best shown by the inoculation of guinea-pigs. 
There are two methods of inoculation, as follows : 

1. The Method of Pfeiffer. — Scrape from the surface of a 
fresh agar-agar culture as much of the growth as will adhere 
to a platinum wire bent into the form of a small loop. Sus- 
pend this amount of material in 1 c.c. of bouillon, and inject 
the suspension into the peritoneal cavity of a guinea-pig 
by means of a hypodermic syringe. With virulent cultures 
this inoculation soon produces a fall in the temperature of 
the animal, which continues and becomes more marked, 
death occurring in from twelve to twenty-four hours. At 
the autopsy of the animal a clear fluid will be found in the 
peritoneal cavity and in the thorax. 

2. The Method of Koch. — This depends upon the fact that 
the animal may be infected through the alimentary canal, 
provided the acidity of the gastric juice be neutralized, this 
acidity being destructive to the cholera spirillum. 

A soft catheter is passed into the stomach of the animal 
through the mouth, and through this 5 c.c. of a 5 per cent, 
solution of sodium carbonate is injected. After ten or fifteen 
minutes 10 c.c. of a bouillon culture of the organism are in- 
jected through the catheter, and immediately afterward the 
animal receives subcutaneously 1 c.c. of the tincture of 
opium for every 200 grams of its body-weight. The object 
of this opium administration is to stop peristalsis, so that the 



PATHOGENIC BACTERIA AND FUNGI. 359 

organisms may be longer in contact with a given area of 
the mucous membrane of the intestine. The result of the 
inoculation first appears after about twenty-four hours. The 
animal then has no appetite and is listless. Later, paralysis 
of the hinder extremities appears, respiration is prolonged 
and weak, the heart-beats become feeble, and the body-tem- 
perature may become subnormal. Death usually occurs 
after the animal has been a few hours in this condition. 

At the autopsy the small intestine will be found to be in- 
jected and containing a flocculent colorless fluid in which 
comma bacilli are present in great numbers. 

Agglutination Reaction. — Cholera spirilla cease their mo- 
tion and aggregate together in clumps, when a small quan- 
tity of the blood-serum of an animal immunized against 
them is added to a suspension of them in salt solution. This 
phenomenon is called the agglutination reaction. 

An agglutinating cholera serum may be produced by in- 
jecting into the ear-vein of a healthy rabbit increasing quan- 
tities of the growth from eighteen-hour agar cultures of 
known cholera spirilla in suspension in normal salt solution ; 
these suspensions must have been heated for one hour at 
6o° C. The quantities and intervals are : first day, 1 loopful 
of the growth ; seventh day, 3 loopfuls ; fourteenth day, 5 
loopfuls ; twenty-first day, about 8 loopfuls. The fourth in- 
jection may be made into the peritoneal cavity and the 
rabbit is ready to be bled on the twenty-eighth day. This 
procedure should give serum which will agglutinate cholera 
spirilla in a dilution of 1 : 4000. The serum should be col- 
lected under precautions to prevent contamination by 
bacteria, and should be kept in small sealed tubes on the 
ice. 1 

Pfeiffer's Reaction. — One loopful of an eighteen-hour cul- 
ture of the cholera spirillum is suspended in 1 c.c. of a dilu- 
tion of agglutinating cholera serum in normal salt solution, 
which is somewhat less dilute than the maximum dilution 

1 These directions are essentially those given by A. J. McLaughlin. Re- 
print from Public Health Reports, No. 53. Public Health and Marine Hos- 
pital Service of the United States. 



360 PATHOLOGICAL TECHNIQUE. 

necessary to agglutinate the cholera spirillum. This mixture 
is injected into the peritoneal cavity of a guinea-pig of about 
200 gms. weight. After about twenty-five minutes some of 
it is withdrawn and examined microscopically, when the 
spirilla will be found to have lost their motility and to have 
become swollen and of degenerate appearance. Ultimately 
they disintegrate and disappear. Other spirilla do not un- 
dergo this change, and the reaction is specific. 

Occurrence. — In the alvine dejections and in the intestinal 
contents of cholera patients (Fig. 105). It apparently only 






*■•* 

V 



^ »» 



Fig. 105. — Cover-glass preparation of a mucous floccule in Asiatic cholera; X 650 

(Vierordt). 

rarely invades the circulating blood. Its presence in the 
vomitus may sometimes be shown. It has been found in the 
water-supplies during epidemics. 

The cholera spirillum is the representative of a large group 
of spirilla, many of which may be found in river waters. 
According to Abbott and Bergey, the only trustworthy 
method of distinguishing some of these from the true chol- 
era spirillum is their failure to manifest a " clump reaction " 
with the serum of an animal immunized to infection with the 
true cholera spirillum. 



PATHOGENIC BACTERIA AND EUNGI. 36 1 

Bacteriological Diagnosis. 1 — Cultures should be made from 
the feces, or contents of the lower end of the ileum, in a special 
fluid medium and on agar Petri plates. Mucus flakes, if possible, 
should be taken for inoculation. The special fluid medium favors 
the growth of spirilla and is prepared as follows : 

Peptone (Chapoteau or Witte) 10.0 

Salt 10.0 

Potassium nitrate .1 

Sodium carbonate . . . .2 

Distilled water 1000.0 

The agar plates are made up with 15 c.c. each of 3 per cent, agar, 
which has been made alkaline by the addition of 3 c.c. of a 10 
per cent, solution of caustic soda to each 100 c.c. of the medium 
after it has been made neutral to litmus. The plates are inoculated 
in sets of three after the agar has solidified by rubbing one loopful 
over the surface of the agar in one plate with a platinum loop or 
a bent glass rod, and then streaking the surfaces of the other 
plates successively with the same loop or rod. The surfaces of the 
solidified agar must be dried before inoculation by placing the 
plates for five minutes in a warming oven at 6o° C, or in the in- 
cubator at 37 C, for one hour, with the covers removed and the 
agar surface downward. The tubes of the fluid medium should 
be inoculated with one loopful of the material and the flasks with 
i c.c. 

After inoculation all cultures are placed in the incubator at 
37° C. 

The uppermost layers of the fluid cultures should be examined 
microscopically after three, six, twelve, and twenty-four hours 
without disturbing the fluid more than is necessary. If spirilla 
are found agreeing in morphology, motility, and staining reac- 
tions with the cholera spirillum, agar plates are to be inoculated 
from this uppermost layer in the same manner as from the original 
material and incubated at 37 C. 

The colonies on the agar plates develop within eighteen hours, 
and appear as pale, semitransparent discs, which show by trans- 
mitted light an opalescent or iridescent quality. Suspicious col- 
onies are to be tested as follows : On a clean glass slide are placed 
at three separate points single drops of a 1 : 200 dilution in phys- 
iological salt solution of an agglutinating cholera serum, such as 
is described on page 192. These drops are numbered on the slide 
1, 2, and 3. With them are then mixed portions of suspicious 
colonies, correspondingly numbered on the slide, by means of a 
straight platinum wire. If the diffuse cloudiness of the drop of 
fluid changes within a few minutes to a clear fluid with flocculi in 
suspension, and the macroscopical and microscopical appearances 

1 A. J. McLaughlin, loc. cit. 



362 PATHOLOGICAL TECHNLQUE. 

of agglutination are produced, the colony is probably that of the 
cholera spirillum. It may be necessary to test in this way numer- 
ous colonies. From colonies thus giving a positive agglutination 
reaction agar slants are inoculated and incubated for eighteen 
hours, when emulsions of the spirilla for more delicate agglutina- 
tion tests are prepared by pouring into each tube 5 to 8 c.cm. of 
sterile physiological salt solution and shaking the tubes. Sus- 
picious colonies not showing agglutination reactions should also 
be planted on agar slants and the growth tested again, because 
freshly isolated cholera spirilla do not always respond to the test. 

The more delicate agglutination tests are carried out as follows : 
In each of a number of small test-tubes of 2 c.cm. capacity is 
placed \ c.c. of dilutions in salt solution of agglutinating serum 
varying from 1 : 10 to 1 : 4000, or up to the limit of the agglutin- 
ating power of the serum. To each tube is then added \ c.c. of 
the emulsion of the suspected spirilla. These manipulations are 
carried out with a pipette, to which is attached a rubber bulb for 
suction and expulsion. The highest dilutions at which agglutina- 
tion appears in the tubes is noted after they have been in the in- 
cubator at 37 C. for one hour, and again after an additional two 
hours at room temperature. If the spirilla are true cholera spi- 
rilla, they will be agglutinated at or near the maximum dilution at 
which the specific serum agglutinates the true cholera micro- 
organism. 

PfeifTer's reaction may also be employed as a confirmatory test. 
Of course the spirilla should also be shown to manifest the other 
characteristics described above before a positive diagnosis is made. 

Dieudonne 's Blood-agar Medium. — This has an inhibiting effect 
on the growth of other micro-organisms than spirilla, and may be 
employed in the same manner as the agar medium described 
above. It is prepared as follows : 

Defibrinated ox blood . 30 

Normal solution of caustic potash 30 

Nutrient agar (3 per cent.) 140 

Add the caustic potash solution to the ox blood and add the 
melted agar. Sterilize for one hour at ioo° C, and use about 15 
to 20 c.c. for each plate. 

The Micro-organism of Actinomycosis. — The proper 
name of this micro-organism is " Actinomyces bovis." It 
belongs to the group of filamentous branching micro-organ- 
isms which are regarded as occupying an intermediate posi- 
tion between the bacteria, on one hand, and the moulds or 
hyphomycetes on the other. 

The organism appears in the pus from subacute or chronic 
suppurative lesions of the disease actinomycosis, as grayish 



PATHOGENIC BACTERIA AND FUNGI. 



363 



or yellowish granules, usually less than 1 mm. in diameter. 
Sometimes these granules are aggregated in groups of two 




Fig. 106. — Actinomyces granule crushed beneath a cover-glass, showing radial 
striations in the hyaline masses. Preparation not stained; low magnifying power 
(Wright and Brown). 

or three, and thus appear as lobulated larger granules. 
They are friable, and when gently crushed beneath a cover- 
glass and observed under the microscope, they are seen to 
have been broken up into hyaline rounded masses, at the 




Fig. 107. — A portion of an actinomyces granule crushed beneath a cover- 
glass, showing the "clubs." The preparation not stained; moderately high mag- 
nifying power (Wright and Brown). 

margins of which, on close inspection, fine radial striations 
or filaments or hyaline club-shaped bodies, all closely set 
together, may be seen (Figs. 106, 107). The club-shaped 



364 



PA THOL O GICAL TECHNIQ UE. 



bodies are variable in size, and are composed of a hyaline, 
refringent substance. The appearance of radial striation in 
the granule, when observed with the microscope, due to the 
presence and radial arrangement of these hyaline bodies, 
gave rise to the name " ray-fungus " for this parasite. Not 
all of the granules have these " clubs." In the granules 
obtained from the lesions in man they are much less fre- 
quently observed than in those obtained from the lesions in 
cattle. 

If a cover-glass preparation be made by breaking up one 
of the granules and staining with Gram's method, there will 
usually be found, upon examination with an oil-immersion 
lens, isolated and matted filaments, many of which may be 




Fig. 108. — Branching actinomyces filaments in a cover-glass preparation 
made from an actinomyces granule stained by Gram's method ; X 1000 (Wright 
and Brown). 

seen to branch, in addition to longer and shorter fragments 
of filaments and fine detritus of the same (Fig. 108). The 
filaments are usually more or less wavy in their course, and 
are, as a rule, slightly thicker than the tubercle bacillus. 
Some of the filaments will be found to stain homogeneously ; 
others do not stain so deeply, and show numerous deeply 
staining points in their substance. If clubs are present in 



PATHOGENIC BACTERIA AND FUNGI. 



365 



the granule, they also may be found scattered throughout 
the preparation. 

In sections of the tissues stained by Gram's method two 
chief forms of granules are found. In one of these forms 
the granule is seen to consist of filaments embedded in a 
hyaline substance, and usually arranged at the periphery 
in an indefinite radiate manner (Fig. 109). At the margin 










Fig. 109. — Colony or granule of actinomyces in a section through a lesion, 
showing the Gram-stained filaments and hyaline material and also the pus-cells 
surrounding the colony (Wright and Brown). 

of the granule the filaments are usually much more numer- 
ous than in the central portions, where the hyaline material 
predominates. This hyaline material apparently consists 
of degenerate or dead filaments or their remains. The 
other form of granule seen in sections is distinguished by 
possessing at its margin a row of closely set radiating club- 
shaped bodies composed of hyaline substance which does 
not stain by Gram's method (Fig. no). These are the 



3 66 



PA THOLOGICAL TECHNIQUE. 



" clubs " previously mentioned, and they may occupy more 
or less of the circumference of the granule. In certain 
instances a Gram-staining filament may be seen in the cen- 
tral portion of a club. The main mass of this form of 
granule is not essentially different from that of the first- 
mentioned form. The characteristics of both forms of 
granule may be found in some granules. 

The club-shaped bodies are to be regarded as products 
of degeneration of the marginal filaments. 

In some cases isolated or small groups of filaments may 
be found scattered among the pus-cells in the lesions. 



* * • 






Sk .♦« ' S>~\.- Y.<. '■ 












:--, 



* 



• • 






i 

* 



Fig. iio. — Colony or granule of actinomyces in a section through a lesion, 
showing the peripheral arrangement of the "clubs." In several instances the 
central stained filaments in the " clubs " are seen ; x 75© (Wright and Brown). 

Diagnosis. — The finding of the granules in suspected pus 
may be facilitated by spreading the pus on a slide. 

The identification of the organism is made certain only 
when the granules have been found to present the appear- 
ances described above after crushing under a cover-glass, 
and after cover-glass preparations made from them and 
stained by Gram's method show the branching filaments. 



PATHOGENIC BACTERIA AND FUNGI. $6j 

Cultures. — Actinomyces bovis is essentially an anaerobe 
and it does not grow at room-temperature. A good growth 
in cultures is obtained only in the depths of solid culture- 
media and in bouillon. Growth is obtained on the surface 
of solid culture-media only when large numbers of the micro- 
organisms are planted upon the culture-media. These sur- 
face growths are white, elevated, more or less nodular, and 
have irregular margins. 

Sugar Agar. — In " stab " cultures and in cultures by the 
method of ' Liborius ' (see page 221), growth occurs only 
below a depth of about 1 cm. from the surface. The colo- 
nies continue to develop during some days in the incubator. 
The larger colonies are spherical, whitish, and may attain a 
diameter of I mm. or more. The smaller colonies, under the 
microscope, are seen to consist of a dense, interlacing felt- 
w T ork of frequently branching filaments, which at the per- 
iphery are disposed in a more or less radiating manner. The 
microscopical colonies may be conveniently studied in thin 
slices cut out of the agar or in frozen sections of the agar 
fixed in formalin and stained by the Gram-Weigert method. 

Bouillon. — : Growth occurs in the form of solid, whitish 
masses in the bottom of the tube ; there is never growth on 
the surface. When first isolated from the lesions the growth 
usually appears in the form of small, nodular, irregular, 
spherical, whitish structures, often adherent to one another, 
and forming mulberry-like masses, but under continued cul- 
tivation most of the strains of the micro-organism finally 
grow in the form of flaky, friable, amorphous masses, which 
in some instances, after some days in the incubator, become 
transformed into a stringy, viscid material. With most strains 
of the micro-organisms the bouillon remains clear. There is 
a good growth in bouillon, without any anaerobic precau- 
tions, apparently because the dense masses in which the 
micro-organism grows furnish sufficient anaerobic conditions 
within themselves. 

Potato.— No growth. 

The production of "clubs" outside of the body may be 
obtained by placing some of the nodular growth from a 



368 PATHOLOGICAL TECHNIQUE. 

bouillon-culture in sterile serum or pleuritic fluid and keep- 
ing it in the incubator for a few days. The filaments of the 
micro-organism in immediate contact with the fluid become 
invested with the hyaline eosin-staining sheath, and the fila- 
ment thus enclosed may no longer stain by the Gram-Wei- 
gert method. In this way structures are produced which 
are identical in every respect with the " clubs " developed 
from the filaments in the lesions. (See Figs. 107, no, in, 

H3-) 

Pathogenesis.— Intraperitoneal inoculation of guinea-pigs 
with suspensions of the growth in bouillon-cultures produces, 
after three or more weeks, with some strains of the micro- 



\ -r\ 




Fig. hi. — Showing " club " formation about the filaments of actinomyces bovis r 
after exposure to the action of serous fluid outside of the animal body. 

organism, granulomatous nodules in the abdominal cavity, 
varying in size up to 1 cm. in diameter. These nodules 
consist of granulation and connective tissue, enclosing small 
abscesses in which are found the characteristic " club-bear- 
ing" colonies or granules. Different strains of the micro- 
organism vary in virulence and some produce no lesions. 

Method of Isolation. — The granules, preferably obtained 
from closed lesions, are first thoroughly washed in sterile 
water or bouillon and then crushed and disintegrated be- 
tween two sterile glass slides. It is well to examine micro- 



PATHOGENIC BACTERIA AND FUNGI. 369 

scopically the disintegrated material to see if filamentous 
masses are present, because in some instances, through de- 
generative changes, the filaments which represent the living 
elements of the granules have died out or disappeared from 
the granules. If no filaments are present, or if they are few 
in number, it is not advisable to proceed further. If, how- 
ever, filaments and filamentous masses are found, then the 
disintegrated products of the granules are to be transferred 
by means of the platinum loop to melted I per cent, dex- 
trose-agar, contained in test-tubes filled to a depth of about 
7 or 8 cm., which have been cooled to about 40 C. The 
material is to be thoroughly distributed throughout the 

•.- 



v^& 



V>* 



Fig. 112. — Small colonies of actinomyces bovis in the depths of an agar culture. 

melted agar by means of the loop, and the tube then placed 
in the incubator. At the same time a number of granules, 
after thorough washing in sterile water or bouillon, should 
be placed in sterile test-tubes, plugged with cotton, and kept 
at room temperature in the dark. 

The sugar-agar tubes, inoculated as above described, 
should be examined from day to day for the presence of the 
characteristic colonies in the depths of the agar. If very 
many colonies of contaminating bacteria develop in the tubes, 
it will probably be very difficult or impossible to isolate the 
specific micro-organism. If there are a few or no contami- 
nating colonies, then the colonies of the specific micro-organ- 

24 



37° 



PA THOL O GICA L TE CffNIQ UE. 



ism should be expected to develop in the course of two or 
three days to a week. If a good number of living filaments 
of the micro-organism have been distributed throughout the 
agar, the specific colonies that develop will be very numer- 
ous in the depths of the agar, especially throughout a shallow 
zone situated about I cm. below the surface of the agar-agar. 
When the presence of the characteristic colonies has been 
determined, slices or pieces of the agar, containing colonies, 
are to be cut out of the tube by means of a stiff platinum 
wire with a flattened and bent extremity. A piece of the 
agar is to be placed on a clean slide and covered with a 
clean cover-glass. It is to be examined under a low power 
of the microscope, and an isolated colony selected for trans- 




>:., a 







Fig. 113. — Portion of a colony of Actinomyces bovis in a section from a lesion 
in a guinea-pig produced by intraperitoneal inoculation. The radiating " clubs " 
at the periphery, some with central filaments, are shown, as well as the felt-work 
of interlacing branching filaments in the central portions. 

plantation. By obvious manipulations, under continuous 
control of microscopic observation, the selected colony, 
together with a small amount of the surrounding agar, is to 
be cut out, care being taken that no other colony is present. 
The small piece of agar thus cut out should not have a 
greatest dimension of more than 2 mm. The piece of agar 
is then transferred from the slide by means of a platinum 
loop to a tube of sterile bouillon, where it is thoroughly 
shaken up in order to free it from any adherent bacteria. If 
there be reason to believe that the small piece of agar has 



PATHOGENIC BACTERIA AND FUNGI. 3?I 

been very much contaminated with bacteria, it should be 
washed in a second tube of bouillon, then the piece of agar 
is to be transferred by means of the platinum loop to a tube 
of melted sugar-agar cooled to 40 C. It should be deeply 
immersed in the agar and the tube placed in the incubator. 
If the colony thus transferred to the agar-agar is capable of 
growth, in the course of some days it will have formed a 
good-sized colony from which transplants in various culture- 
media may be made. 

In the manner described several small pieces of agar con- 
taining single isolated colonies should be placed in sugar- 
agar tubes, because the chances are that some of the colonies 
will not grow, and contaminations with other bacteria may 
occur. 

If the number of contaminating colonies is so great in the 
original agar-cultures from the granules that it is found im- 
possible or very difficult to obtain specific colonies free from 
other micro-organisms, then it is probably not worth while 
to expend much labor with the task of isolation from these 
original agar-tubes, but it is much better to wait until the 
granules placed on the sides of sterile test-tubes have dried 
thereon for two or three weeks, and then proceed with these 
granules as just described for the fresh granules. The dry- 
ing of the granules for this length of time will probably 
suffice to kill off most of the contaminating bacteria and 
enable isolated colonies of the specific micro-organism to be 
obtained in the agar suspension-cultures. 

To Stain the Actinomyces in Sections. — In staining 
the actinomyces it is important to stain not only the fila- 
ments and other forms of the organism but also the hyaline 
swollen sheaths which surround the ends of the filaments. 
Eosin followed by methylene-blue sometimes gives good 
results. Good preparations can also be obtained by staining 
in alum-hematoxylin, followed by a strong solution of eosin ; 
place the sections for five to thirty seconds in acid alcohol, 
and then wash thoroughly in water before dehydrating in 
alcohol. It is believed that the two following methods will 
give better results than can be obtained by any of the 



372 PATHOLOGICAL TECHNIQUE. 

methods previously published for this purpose. The first is, 
perhaps, the better and surer, although the clubs are some- 
times brought out more intensely by the second method. 

Formaldehyde and alcohol fixation are preferable to 
Zenker's fluid for the study of this micro-organism, but not 
for the study of the lesions produced by it. 

Mallory's Stains. — Method No. 1. — i. Stain sections 
deeply in a saturated aqueous solution of eosin for at least 
ten minutes. 

2. Wash off in water. 

3. Stain in aniline-methyl-violet two to five minutes. 

4. Wash off with normal salt solution. 

5. Iodin solution (1:2: 100) one minute. 

6. Water. Blot with filter-paper. 

7. Aniline oil until section is clear. 

8. Xylol, several changes. 

9. Xylol balsam. 

A light preliminary stain with alum-hematoxylin will often 
be found useful to bring the nuclei out sharply. 

Method No. 2. — r. Stain lightly in alum-hematoxylin 
three to five minutes. 

2. Wash in water. 

3. Dehydrate in 95 per cent, alcohol. 

4. Fasten section to slide with ether-vapor. 

5. Aniline-methyl-violet five to twenty minutes. 

6. Wash off with water. 

7. Dry with filter-paper. 

8. Aniline saturated with fuchsin one to three minutes. 

9. Wash out the fuchsin with pure aniline until the clubs 
are sharply differentiated : watch the process under the low 
power of the microscope. 

10. Xylol, several changes. 

11. Xylol balsam. 

The polymorphous bacterium is stained blue, the swollen 
membrane (the club), light to dark pink. By these methods 
it is possible to demonstrate in sections containing young 
colonies the ends of the threads stained blue surrounded by 
the swollen cell-membrane stained pink. 



PATHOGENIC BACTERIA AND FUNGI. 373 

Sporotrichum Schenckii. — This is a fungus or hypo- 
mycete which is believed to be the infectious agent in sporo- 
trichosis, a disease characterized by the formation of 
gumma-like nodes, abscesses, and ulcers chiefly involving 
the skin and subcutaneous tissue. 

Morphology. — In the lesions of the spontaneous disease 
the fungus elements are difficult or impossible to distinguish 
from cellular detritus, apparently because they exist therein 
■ in certain small spore-like forms. 

In culture the fungus appears in the form of a mycelium 
composed of branching septate filaments with abundant 
formation of spores. The filaments vary somewhat in thick- 
ness, their average being about 2 microns, have refringent 
walls, refractive granules in their interior, and transverse 
septa at fairly regular intervals. The spores grow singly 
along the sides of the filaments and in clusters of from 3 to 
6 or more at the ends of filaments, which may be slightly 
expanded. They are ovate or spiculate bodies, 3 to 5 
microns in their longest diameter, having a granular interior 
and a double contour. They are attached to the filaments 
by delicate pedicles, which are easily broken. With basic 
aniline dyes and with Gram's method of staining they stain 
generally evenly, but sometimes show vacuole-like areas. 
The filaments do not stain as deeply as the spores and may 
show in their interior more deeply staining granules. 

Cultures. — The fungus grows on all of the usual culture- 
media, best in those containing sugar. It is aerobic, and 
thrives as well at room temperature as in the incubator. 
The colonies on solid media are at first pale gray or nearly 
white, and have a delicate fringe of radiating filaments at 
their margins. Later, they spread widely over the surface 
of the media and form a layer or a membrane 1 or more 
mm. thick with wrinkled surface, which may become brown 
or black in color and of a velvety appearance. Down-growth 
may occur into the underlying media. In stab cultures, 
lateral outgrowths occur along the line of inoculation. Gela- 
tine is slowly liquefied by the micro-organism. In bouillon, 
growth appears as a downy sediment. The growth in the 



374 



PA THOL O GICAL TE CHNIQ UE. 



original cultures from the lesions may be slow, the colonies 
appearing after a week or more. In succeeding generations 
the growth is more rapid. 





• 

■4. * * . **»* 




. % * « 
*, & -** "1 


*• 

• 


& -- * i. 



Fig. 114. — Sporotrichum Schenckii. Section of nodule in abdominal wall of 
white rat. Gram's stain. Cells and spores, the latter oblong and deeply colored ; 
X about 1000 (L. Hektoen and C. F. Perkins). 




Fig. 115. — Sporotrichum Schenckii. Colonies on glycerin-agar plate. Low 
power (L. Hektoen and C. F. Perkins). 

Pathogenesis. — The micro-organism is pathogenic for ex- 
perimental animals, especially for white rats. In the latter 



PATHOGENIC BACTERIA AND FUNGI. 375 

animals, septicemia, and disseminated suppurative or granu- 
lomatous lesions may be produced by inoculation with cul- 
tures. The testicles are very frequently involved after 



1 



Fig. 116. — Sporotrichum Schenckii. Margin of hanging-drop culture ; X about 
150 (L. Hektoen and C. F. Perkins). 




Fig. 117. — Sporotrichum Schenckii. Margin of hanging-drop culture ; X about 
1000 (L. Hektoen and C. F. Perkins). 

intraperitoneal inoculation. In the experimental lesions the 
organism does not appear in the filamentous form, but only 
in modified forms, more or less resembling the spores and 



376 PATHOLOGICAL TECHNIQUE. 

as oblong Gram-staining bodies, I to 3 microns wide and 10 
to 12 microns long. Sometimes small bud-like processes 
project from these forms. 

Occurrence. — The fungus is thought to have a natural 
habitat in the outer world and to be widely distributed. It 
is claimed that spontaneous infection with it has been ob- 
served in the dog and rat, and that some of the cases of 
epizootic or mycotic lymphangitis in horses are due to infec- 
tion with it. 

Diagnosis. — The only practical way by which Sporo- 
trichum Schenckii may be recognized in suspected sporo- 
trichosis is by obtaining it in cultures from unopened gum- 
mata or abscesses. As has been pointed out above, it is 
practically impossible to recognize it in lesions by direct 
examination. 

The Blastomycetes. — Under this heading are included 
those pathogenic fungi which are regarded as the infectious 
agents of the granulomatous and suppurative processes, 
known under the names of blastomycosis, oidiomycosis, 
blastomycetic dermatitis, coccidioidal granuloma, and certain 
others. 

Morphology. — In the lesions the micro-organisms appear 
generally as spherical bodies, each consisting of a proto- 
plasmic mass enclosed in a double-contoured hyaline cap- 
sule. The diameter of the bodies varies up to 30 microns 
or more. In the protoplasm vacuoles, granules, and various 
markings may be seen, but no nucleus is apparent. The 
mode of proliferation in the lesions in the majority of cases 
is by gemmation or budding. The micro-organisms in some 
cases have great resemblance to yeast fungi or saccharo- 
myces. In the minority of the known cases proliferation of 
the micro-organisms in the lesions is not by budding, but by 
a process which is regarded as one of sporulation, the proto- 
plasm of the larger forms segmenting into many small 
spherical bodies. Each of these small spherical bodies ac- 
quires a capsule and, being set free by the rupture of the 
capsule of the mother-cell, develops into an adult parasite. 
In the case reported by E. B. Wolbach, the hyaline cap- 



PATHOGENIC BACTERIA AND FUNGI 



377 







► : . # ^j 


,fe ^%.'4* : 


Jfcl&t *. 




"'"■^ ...... .... 




teL 


'7»SS 


*<. ^. .w - 



Fig. 118. — Blastomycetes in a giant-cell in the lung. Two of the organisms are 
budding; X about iooo (Mallory). 




sule of the micro-organism was provided with prickles and 
spines. 



378 



PA THOL O GICA L TE CHNIQ UE. 



Cultural Peculiarities. — The micro-organisms grow well at 
room temperature and in the incubator and upon any of the 
usual culture-media. According to H. T. Ricketts, the micro- 




Tic. 120.— Organism of "Dermatitis Coccidioides." Edge of seventy-two-hour 
agar colony ; X 260 (S. B. Wolbach). 




FiG. 121. — Organism of " Dermatitis Coccidioides." Sprouting sphere at end 
of twenty-four hours' incubation, showing the development of filaments from it; 
X 400 (S. B. Wolbach). 

organisms obtained from various cases may be divided into 
three groups, according to their biological characters, as 
shown in the cultures as follows: 

Group 1. — Those growing chiefly as spherical or oval 



PATHOGENIC BACTERIA AND FUNGI. 



379 



budding cells and resembling the yeasts, but capable of pro- 
ducing mycelium. The colonies on solid media are elevated, 
soft, moist, and white, coalescing to form a fleshy growth of 




mm. 4 ' 




Fig. 122. — Organism of "Dermatitis Coccidioides." Various stages in the 
development of the organism as seen in tissue (Mallory). 

paste-like consistence. Microscopically, the colonies are 
finely granular. In fluid media, growth appears as a floccu- 
lent sediment in a clear supernatant fluid. 

Group 2. — Those forming a submerged mycelium which 
break up into chains of spores, while proliferation by bud- 
ding is not a prominent feature. The colonies at first are 



380 . PATHOLOGICAL TECHNIQUE. 

granular and slightly elevated. Later they coalesce, become 
more elevated, and incorporate themselves in the medium. 
Eventually they form a surface like that of a piece of 
crumpled cloth. Microscopically, the colonies appear as 
masses of radiating, segmented, and branching hyphae. In 
fluid media growth appears as a membrane at the surface 
with coherent masses or tufts at bottom and sides of the tube. 

Group 3. — Those producing mycelium with fruit-bearing 
aerial hyphae, and also capable of multiplying by gemmation 
or budding. The colonies are dry, white, and feathery in 
appearance, develop hyphae, which eventually cover the inner 
surface of the tube. There is growth into the media. Micro- 
scopically, the growth is made up of branching, closely seg- 
mented hyphae with many lateral off-shoots. In fluid media 
the growth appears as a coherent mycelial tuft at the bottom 
of the tube with no surface growth. 

The micro-organisms of the first and second groups are 
capable of producing fermentation, while those of the third 
group are not. 

Pathogenesis. — In animals inoculated with cultures of some, 
but not all, of these micro-organisms, abscesses, granuloma- 
tous tumors, and tubercle-like nodules, widely disseminated, 
have been produced. In these lesions in the animals the 
micro-organisms exist in the same form as in the human 
lesions. Wolbach, working with the micro-organism of the 
type which proliferates in the tissues by sporulation, and 
which was not observed to proliferate by budding in cultures, 
has traced in animals the transformation of the hyphae of the 
cultures into the characteristic spherical bodies of the lesions. 
He found that the spherical bodies arise by the segments of 
the hyphae, enlarging and assuming spherical shape, the wall 
of the segment thus becoming the capsule of the spherical 
body. He also observed pointed and club-shaped hyaline 
bodies radiately arranged at the periphery and continuous 
with the capsules of the micro-organisms. 

Diagnosis. — The micro-organisms may be easily recog- 
nized by mixing a drop of the pus or granulation tissue from 
a lesion with a little 10 per cent, solution of sodium hydrate 
and examining the mixture under a cover-glass with the 
microscope. 



ANIMAL PARASITES. 



I^ntamcebse. — At least two species of amebae occur in 
the intestine, entamoeba coli and histolytica. The first is a 
harmless parasite ; the other may give rise to amebic dys- 
entery. 

In cases of dysentery suspected of being due to amebae 
the stools are best examined as soon as voided, although the 
amebae will sometimes remain active in stools even over 
twenty-four hours old. A warm stage during the examina- 
tion is an advantage, but not a necessity. A drop of the 
fluid material, preferably that containing mucus or blood, is 
placed on a slide and lightly covered with a cover-glass. If 
the slide is cold and the organisms do not move, warm the 
slide gently and the movements of the amebae will often 
start up. Pus from abscesses due to the amebae is examined 
in the same way. A positive diagnosis rests on the presence 
of the characteristic large, pale cells, consisting of nucleus, 
granular endosarc, and hyaline ectosarc, and on the move- 
ments of the protoplasm, which projects itself more or less 
actively in the form of pseudopodia. 

Smear preparations made from abscesses or feces can be 
stained in a variety of ways to show the amebae, for example, 
by Wright's or Giemsa's method or by iron hematoxylin. 
The simplest way is to fix the preparations for a few minutes 
in Zenker's fluid, wash in water, and stain with alum-hem- 
atoxylin. Delafield's solution is highly recommended. 

In sections of fixed tissues the nuclei of the amebae do not 
stain particularly well with the ordinary nuclear stains, such 
as alum-hematoxylin and methylene-blue, although phos- 
photungstic acid hematoxylin brings them out with great 
sharpness. The following method of staining them has been 
found to give very satisfactory results and to render the 
recognition of the organisms easy: 

381 



3^2 PATHOLOGICAL TECHNIQUE. 

Differential Stain for the Entameb'ce {Mallory). — I . Harden 
in alcohol. 

2. Stain sections in a saturated aqueous solution of thionin 
three to five minutes. 





™ i 



1 






Fig. 123. — Entamoeba histolytica in section of ulcer of intestine ; X 500 (photo 
by F. B. Mallory). 

3. Differentiate in a 2 per cent, aqueous solution of oxalic 
acid for one-half to one minute. 

4. Wash in water. 

5. Dehydrate in absolute alcohol 

6. Clear in xylol. 

7. Xylol-balsam. 

The nuclei of the amebae and the granules of the mast- 
cells are stained brownish red ; the nuclei of the mast-cells 
and of all other cells are stained blue. 



ANIMAL PARASITES. 



383 



Entamoeba 

coli, 
Schaudinn, 

1903. 

Entamoeba 
histolytica, 
Schaudinn, 
1903. 


p 
3 
a 


Ten to 30 

microns, 

generally 

smaller 

than 

entamoeba 

histolytica 

or 

entamoeba 

tetragena. 

Ten to 70 
microns, 
generally 
from 15 to 
40 microns. 


9 


Small, blunt, 
and not clear- 
ly differentia- 
ted from rest 
of parasite. 

Blunt or slen- 
der and finger- 
shaped. Very 
refractive and 
clearly differ- 
entiated from 
rest of the 
parasite 


2 

a. 



O 


Sluggish. 
Active. 


O 


Ectoplasm not dis- 
tinct, except when 
moving, and then 
only because it is 
free from granules. 
Is grayish in color 
and not very re- 
fractive. Endoplasm 
is gray, finely gran- 
ular, few non-con- 
tractile vacuoles. Is 
not generally pha- 
gocytic for red 
blood-corpuscles. 
Ectoplasm is very 
distinct and refrac- 
tive, in some in- 
stances even when 
motionless. Glassy 
appearing. Endo- 
plasm is granular, 
contains numerous 
non-contractile vac- 
uoles and red blood- 
corpuscles, when 
latter are present 
in feces. 


3 



p 
3 


Distinct, 
having a 
well-defined 
nuclear 
membrane 
and much 
chromatin. 
Large kary- 
osome. 

Indistinct. 
No well-de- 
fined nu- 
clear mem- 
brane and 
but little 
chromatin. 
Minute 
karyosome. 


c 

(0 


Present. Eight 
young amebae 
developed with- 
in cyst. 

Minute spores 
developed by 
budding meas- 
ure 3 to 5 mi- 
crons. Possess 
a resistant mem- 
brane like a cys- 
tic covering. 
Development 
of the spores 
have not been 
studied. 




v; 
(/) 

o* 

3 
p 

0' 
3 


Doubtful. 
Doubtful. 


O 

c 
ft 
V 


By simple divi- 
sion; autogenous 
sexual reproduc- 
tion in cyst ; and 
by schizogomy 
with the produc- 
tion of eight 
daughter amebas. 
Eight amebas are 
produced within 
the cyst. 

By simple divi- 
sion ; gemmation ; 
and by the bud- 
ding of chromidial 
massessurrounded 
by protoplasm 
from the periph- 
ery of the mother 
parasite, forming 
minute spores. 


II 

5 O 
? 


Is not patho- 
genic, occur- 
ring in a large 
percentage of 
healthy indi- 
viduals. 

Is the cause 
of a form of 
amebic dys- 
entery. 


p 

Er 
■ 

CK5 
ft 

3 
ft 


WithWright's 
stain, ecto- 
plasm, light 
blue ; endo- 
plasm, dark 
blue; and 
nucleus red. 

WithWright's 
stain, ecto- 
plasm, dark 
blue ; endo- 
plasm, light 
blue ; and 
nucleus, pale 
red or pink. 


W 

5' 

5' 

OK? 



3 84 PATHOLOGICAL TECHNIQUE. 

Excellent results were obtained by this method with bits 
of the purulent discharge from a so-called amebic abscess 
of the liver. After hardening in 95 per cent, alcohol, small 
fragments the size of a pin-head and less were stained as 
above directed, and teased apart after they were in the 
balsam. The reddish nuclei stood out so sharply in the 
bluish background of fragmented nuclei and granular de- 
tritus that they were easily picked out with the high dry 
power. 

The results obtained with feces examined in the same way 
or after imbedding in celloidin were much less satisfactory, 
for the reason that various substances in the feces precipitate 
the thionin in the form of reddish crystals and give rise to 
deceptive pictures. A similar differential stain can be ob- 
tained by Unna's method for staining the granules of mast- 
cells (see page no). 

Other protozoa, such as the cercomonas and trichomonas, 
are best examined in fresh preparations. 

The Cultivation of Entamcebse. — The method of W. E. 
Musgrave and M. T. Clegg is as follows : Make Petri plate 
cultures on nutrient agar by streaking the surface of the 
medium with the material containing amebae. The agar 
should be I per cent, alkaline to phenolphthalein, and should 
be of the following composition : 



Agar, 


20; 


Sodium chloride, 


3; 


Beef extract, 


3; 


Water, 


1000. 



The material containing amebae should be prepared by 
placing it in sterile flasks, adding to it 1 c.c. of alkaline bouillon 
to each 100 c.c, and setting it aside for twenty-four to forty- 
eight hours. A loopful of the material from the surface should 
betaken for the inoculation of each Petri plate. The plates are 
kept at a temperature not over 37 C. The temperature 
should be so regulated that the bacteria do not grow so pro- 



ANIMAL PARASITES. 385 

fusely as to interfere with the growth of the amebse. After 
two or three days, if growth of amebae has occurred, trans- 
plant to fresh plates. 

Cultures of the amebae containing only one kind of bac- 
teria may be obtained in the following manner : With a loop, 
infected with a pure culture of the bacterium with which it is 
desired to cultivate the amebae, make several concentric 
ring-shaped inoculations of different diameters on a sterile 
agar plate, then inoculate the center of the plate with the 
mixed bacteria and amebae culture and incubate. After twenty- 
four to forty-eight hours the amebae will have multiplied and 
wandered out over the surface of the agar to the periphery of 
the plate, passing on their way through the rings of growth of 
the pure culture of the bacteria, whereby they tend to lose their 
mixed bacterial content and take up the bacteria of the pure 
culture. Amebae which have passed the outer ring of bacterial 
growth are to be transplanted to the center of fresh plates in- 
oculated with a pure culture, as above described. This is 
repeated until a plate culture containing a pure culture of the 
bacterium is obtained. 

Malarial Organisms. 1 — Three varieties of the plasmodium 
malariae have been described — namely, the tertian, quartan, 
and estivo-autumnal parasites. They develop within or upon 
the red corpuscles and cause the destruction of the corpuscles 
affected. The earliest forms of the parasite appear in the 
blood during the latter part of the malarial paroxysm or 
shortly after it. At this time they appear as small, color- 
less, disc-shaped hyaline bodies which occupy but a small 
portion of the blood-corpuscles. They possess a varying 
degree of ameboid movement, the amount depending upon 
the type of the organism. These ameboid movements are 
best observed on the warm stage. During the process of 
development the parasites increase in size and more or less 
completely fill the red corpuscles containing them. Small 

1 For some important details, here omitted, concerning the morphology and 
biology of the malarial parasites the reader is referred to the authoritative papers 
by Mary Rowley- La wson, Jour. Exp. Med., xiii., p. 263, and by Charles F. 
Craig, Osier's Modern Medicine, i., p. 392. 
25 



386 PATHOLOGICAL TECHNIQUE. 

particles of reddish-brown pigment are produced, during 
their growth, from the hemoglobin of the corpuscles in 
which the organisms are developing. These granules show 
varying degrees of motion, probably imparted to them by 
the movements of the parasites. At first the pigment ap- 
pears to be scattered about in the corpuscle, but it is in real- 
ity in the extremities of the pseudopodia. Later it appears 
more evenly spread about in the periphery. Toward the 
end of the cycle of development the pigment collects in the 
center of the parasite ; at this time the ameboid movements 
have ceased, indications of segmentation occur, and the 
parasite nearly or completely fills the corpuscle. Oftentimes 
at this stage only a small portion of the corpuscle is visible 
at some point on the edge of the parasite. 

The beginning of segmentation is indicated by a number 
of radial lines extending from the periphery of the parasite 
toward the central clump of pigment. Segmentation takes 
place, and the pigment is surrounded by a number of dis- 
tinct segments which vary with the type of the organism. 
Each of these segments shows a central refractive spot 
which probably is the nucleus. At this time one notices 
small hyaline bodies, like those of the early stage in the de- 
velopment of the parasite, in some of the red blood-cor- 
puscles. Oftentimes such a regular process of segmentation 
is not observed, but enough has been said to indicate the 
manner in which reproduction occurs. Segmentation is the 
indication of an approach of a paroxysm. Extra-cellular 
forms of the parasites are not infrequently seen. They may 
be fully grown organisms which have destroyed the cor- 
puscles that contained them, or they may be partly grown 
organisms which have left the corpuscles. These free para- 
sites are indistinct in outline and contain pigment. They 
possess ameboid movements, and may be considerably larger 
than a red blood-corpuscle. Various changes are observed 
in them : 

I. They may increase in size until they become nearly as 
large as polymorphonuclear leucocytes. With the increase 
in size there is a gradual cessation in the movement of the 









?V 



km 



w 




Tertian malarial parasites in red blood-corpuscles (Wright's stain) : i, Young 
parasites (in the corpuscle on the right two or three parasites) ; 2, young parasites ; 
3, half-grown parasites; 4, half-grown parasite (on the left, a blood-plate and near 
the center another blood-plate lying on a red corpuscle) ; 5, half-grown parasites 
(in the corpuscle on the left two parasites) ; 6, full-grown parasite (the nucleus lies 
in a clear space). All the infected blood-corpuscles in the foregoing figures contain 
minute granules that stain red (granular degeneration) (photos by L. S. Brown). 




Two stages in the process of segmentation of a tertian malarial parasite in 
a red blood-corpuscle. Preparation of fresh blood, not stained (Wright and 
Brown). 





« 



jgf 





Tertian malarial parasites in red blood corpuscles (Wright's stain) : i, Young 
parasite ; 2, young parasite with blood plate at the margin of the corpuscle; 3, 
two young parasites in one corpuscle ; 4 and 5, immature parasites ; 6, immature 
parasite (on the left two blood plates); 7, adult parasite (chromatin of nucleus 
in cl :-ar space) ; 8 and g, adult parasites (the chromatin is the reticular mass 
near the center of the parasites) ; 10, segmenting parasite (the chromatin has 
divided into a number of separate dark colored masses. In the center some 
pigment). In all of the foregoing figures, except Figure i, the granular degen- 
eration of the infected red corpuscles is shown (photos by L. S. Brown). 



ANIMAL PARASITES. 387 

pigment-granules, until finally the organisms present the ap- 
pearance of misshapen masses of protoplasm containing 
motionless pigment-granules. 

2. They may undergo fragmentation and give off several 
small circular pigmented bodies. 

3. Vacuolization may occur. 

4. Flagellate forms may develop. One or more thread- 
like processes are thrust out from the organisms. These 
flagella may contain pigment, and may break away from the 
organism and move about among the corpuscles, looking not 
unlike the spirilla of relapsing fever. 

The three varieties of parasites differ from one another in 
a number of ways. The chief differences are the length of 
the cycle of development ; the size of the full-grown organ- 
isms ; the difference in the refractibility of the organisms ; 
the quantity, size, and color of the pigment-granules ; the 
degree of ameboid movement; and the number and shape 
of the segments into which the full-grown organisms divide. 
In the earliest stage the varieties or organisms cannot be dis- 
tinguished from each other. 

The tertian parasite completes its cycle of development in 
about forty-eight hours. When it has attained its fullest 
growth it almost fills the corpuscle, which has become 
larger than normal. This organism is less refractive than 
either of the other two. The pigment-granules are more 
numerous, finer, and more reddish-brown in color; the ame- 
boid movements are much more active ; the segments are 
more irregular in shape and more numerous than those of the 
quartan parasite, varying from twelve to twenty in number. 

The quartan appears to complete its cycle of development 
in from sixty-four to seventy-two hours. The full-grown 
organism does not fill completely the corpuscle, and the latter 
is not increased in size. The organism is more refractive 
than the tertian parasite. The pigment-granules are fewer 
in number, coarser, and have a darker-red color. The ame- 
boid movements are slower ; the segments are pear-shaped, 
more symmetrical, and' less numerous than those of the ter- 
tian parasite, varying from six to twelve in number. Seg- 



388 



PA THOL O GICAL TE CHNIQ UE. 



mentary organisms are more numerous in the peripheral 
circulation than in the case of the tertian parasite. 

The estivo-autumnal parasite cannot be studied so thor- 
oughly in the peripheral ci rculation, because the latter develop- 
ment and segmentation take place in the internal organs. The 





Fig. 124. — Estivo-autumnal malarial parasites in red blood-corpuscles 
(1, 2, 3, and 4, Wright's stain): i, 2, and 4, young parasites; 3, on the left, a 
" crescent," on the concave side of which is shown a portion of the periphery 
of the red corpuscle, which it distends ; on the right, a young parasite ; 5 and 7 
"crescents" in red blood-corpuscles; 6, ovoid form of parasite in a red blood- 
corpuscle (photos by L. S. Brown). 



length of time required to complete its cycle of development 
is not so definitely settled. It appears to require from twelve 
to twenty-four hours, more or less. The full-grown organ- 
ism is smaller than the tertian parasite, and the corpuscle 
which contains it is often smaller than normal and more or 



ANIMAL PARASITES. 389 

less distorted. The parasite is quite refractive. The pig- 
ment-granules are few in number and coarse. The ameboid 
movements are slow. After the duration of fever for from 
five days to a week or more, elongated, ovoid, or crescent- 
shaped bodies make their appearance. They are sometimes 
as large or larger than a red corpuscle. These bodies are 
not a result of segmentation, but appear to be a further de- 
velopment of the round hyaline bodies. They are highly 
refractive and contain granules of coarse pigment in the 
center. They lie usually at one side of the red corpuscles, 
the latter more or less completely filling the concavity be- 
tween the two horns of the crescent. They may lie in the 
center of the corpuscles. Some of the apparently free ovoid 
bodies are turned in such a way as to present a convex sur- 
face toward the observer. 

Double infections occur quite frequently in both tertian 
and quartan fever, and in the latter not infrequently triple 
infections occur. In the double infections two groups of 
parasites reach maturity on successive days and cause daily 
febrile paroxysms. In the triple infection of quartan fever 
three groups of organisms mature on successive days and 
cause corresponding paroxysms. 

Methods of Examining the Blood for Malarial Organisms. 
■ — The organisms of malaria can be detected in fresh speci- 
mens of blood or in specimens of blood which have been 
fixed and stained. 

In doubtful cases the parasites are more surely and easily 
found in cover-glass preparations of the blood fixed and 
stained by special methods. 

The method employed in making cover-glass preparations 
of the blood has been thoroughly described (see preparation 
of cover-glass specimens in the Examination of the Blood, 
page 415). 

Wright's stain for malarial parasites is identical with his 
blood-stain and is applied in the same way (see page 418). 
It gives the so-called Romanowsky stain to the parasites. 

Note. — The description of the development of the parasites is abstracted 
from Thayer and Hewetson' s The Malarial Fevers of Baltimore. 



39° PATHOLOGICAL TECHNIQUE. 

With the stain the body of a malarial parasite stains blue, 
While the color of the chromatin varies from a lilac color 
through varying shades of red to almost black. In the 
young forms of the tertian and estivo-autumnal parasites the 
chromatin appears as a spherical, very dark-red body, while 
in the older forms of the tertian parasite it has a more lilac 
or purplish-red color, and may appear in the form of a 
reticulum. In the intermediate forms the color of the chro- 
matin may present variations between these extremes (see 
Fig. 124). 

Blood plates apparently situated within red blood-cor- 
puscles may be mistaken by the inexperienced for young 
malarial parasites. This ought never to occur if one bears 
in mind the fact that the young parasite of all the three 
kinds should present by this method a dark-red, spherical 
nucleus and a homogeneous blue cytoplasm which is usually 
in the form of a definite ring (see Fig. 124). 

Various workers have shown by their modifications of the 
Romanowsky method that red blood-corpuscles harboring 
malarial parasites have dark-red staining granules. These 
granules may be brought out by the present method, but in 
order to bring them out, it may be necessary to allow the 
staining fluid, after the addition of the water to it, to remain 
on the preparation for at least five minutes, and then not to 
decolorize or differentiate with water for as long a time or to 
such an extent as for ordinary blood preparations. 

In examining a fresh specimen of the blood for the malarial 
organisms a glass slide is substituted for one of the cover- 
glasses, and the cover-glass which has the drop of blood on 
its surface is dropped lightly upon the glass slide and allowed 
to remain there. The first four or five drops of blood 
should be quickly wiped away from the ear until a very 
small drop is obtained. Great care must be exercised to 
touch only the tip of the drop with the cover-glass, so as to 
avoid smearing the blood. If the blood is smeared on the 
cover-glass, the edges of the blood-drop will dry before the 
cover-glass can be transferred to the slide, and the blood will 
not spread. It is necessary that the blood should spread in 



ANIMAL PARASITES. 39 1 

a thin layer in order to study satisfactorily the individual 
corpuscles. If one desires to study the preparation for 
several hours, the edges of the cover-glass can be sur- 
rounded by melted paraffin or vaselin to exclude the air. 
The examination should be made with an oil-immersion lens. 
It should be remembered that the action of cold inhibits the 
ameboid movements of the parasites ; it may be necessary, 
therefore, at times to warm the slide before examining the 
specimen. Evaporation not infrequently occurs, caused by 
the air penetrating beneath the cover-glass. This produces 
changes in many of the corpuscles which may be mistaken 
for hyaline bodies : the central depression becomes paler and 
less refractive than the periphery of the corpuscles ; later a 
number of corpuscles contain small glistening points, and 
still later the corpuscles become crenated. 

GiemscCs Stain. — This also gives the Roman owsky stain- 
ing. The formula is as follows : 

Azur II. — eosin, 3 gm. ; 

Azur II. 0.8 " 

Glycerin (Merck, chemically pure), 250 " 

Methyl-alcohol (Kahlbaum I.), 250 " 

The staining fluid is manufactured by Griibler, and it is 
best to obtain it already prepared. 

1. The preparation is dried in the air and fixed in absolute 
alcohol fifteen minutes, or in methyl-alcohol two or three 
minutes, after which the alcohol is . removed with filter- 
paper. 

2. To I c.c. of distilled water in a small graduate add 1 
drop of the staining fluid and shake gently. This dilution is 
to be made immediately before proceeding to the next step. 

3. Cover the preparation with freshly diluted staining fluid 
for ten to fifteen minutes. 

4. Wash in a stream of water. 

5. Remove excess of water with filter-paper, dry in the 
air, and mount in balsam. 

If specially intense staining is desired, add to the distilled 



392 PATHOLOGICAL TECHNIQUE. 

water before mixing it with the stain a little potassium car- 
bonate solution in the proportion of I or 2 drops of a I per 
cent, solution to 10 ex. of water. 

Giemsa's Method for Staining Protozoa and Bac- 
teria in Sections. — I. Fix pieces of tissue not more than 
2 mm. thick in sublimate alcohol, consisting of 2 parts of a 
concentrated aqueous solution of corrosive sublimate and I 
part of absolute alcohol. The fixation requires at least forty- 
eight hours. The fixing fluid is to be renewed after twenty- 
four hours. 

The tissue may remain for as long as three months in the 
fixing fluid without disadvantage if evaporation is prevented. 

2. Dehydrate in graded alcohols and xylol. Embed in 
paraffin. The sections should not be over 4 microns thick — 
two microns are better. The tissues must not be handled 
with metal instruments until after they have been cleared in 
oil of cedar wood. 

3. Treat sections with xylol, followed by graded alcohols 
and water. 

4. Ten minutes in a solution consisting of KI, 2 gms. ; 
distilled water, 100 c.c. ; Lugol's solution, 3 c.c. 

Instead of this mixture, it is possible to use Lugol's solu- 
tion only (1 to 3 c.c. of it mixed with 100 c.c. of water or 70 
per cent, alcohol), or tincture of iodin diluted with alcohol. 
The use of the weak alcoholic iodin solution is indicated 
when a more intense blue staining of the cytoplasm is de- 
sired. Treatment with the weaker iodin solutions demands 
naturally a longer time — twenty to thirty minutes. 

5. Treat with 95 per cent, alcohol until the yellow color 
is removed. After a quick wash with distilled water place 
sections for ten minutes in a 0.5 per cent, aqueous solution 
of sodium hyposulphite, then five minutes in tap-water, and 
for a short time in distilled water. 

6. Stain with freshly diluted Giemsa solution two to twelve 
hours or longer. The solution recommended for this pur- 
pose should be made up according to the following modi- 
fied formula: 



ANIMAL PARASITES. 393 

Azur II. — eosin, 3 gm.; 

Azur II. 0.8 " 

Glycerin, 125 c.c. ; 

Methyl-alcohol, 375 " 

The dilution should be 1 drop to 1 c.c. of water; or for 
a longer period of staining, 1 drop to 2 c.c. of water. After 
the first half hour the staining mixture is to be poured off 
and replaced by fresh. 

7. Wash in distilled water and dehydrate as follows : 
(a) Acetone 95 c.c. plus xylol 5 c.c. 

(J?) Acetone 70 c.c. plus xylol 30 c.c. 

(c) Acetone 70 c.c. plus xylol 30 c.c. 

(d) Xylol pure. 

(e) Cedar oil. 

8. Mount in cedar oil. 

The duration of the treatment with a, b, and c depends 
upon the degree of differentiation required. 

The distilled water used for diluting the staining fluid 
must be absolutely free from acid. The slightest trace of 
organic or mineral acids, or even the presence of a consider- 
able amount of carbonic acid, spoils the staining. The dis- 
tilled water may be tested and corrected for use as follows : 

Place 300 c.c. of it in each of 4 flasks. Add 1 per cent, 
solution of carbonate of sodium (Na 2 C0 3 ), I drop to first 
flask, 2 drops to second flask, and so on. Then take 10 c.c. 
from each flask in a clean test-tube and add 2 or 3 drops of 
a fresh solution of hematoxylin in absolute alcohol, which 
should be pale yellow to nearly colorless. Stand against a 
white background, and that flask with the right reaction 
should take on a violet tinge after one to five minutes. 

For bringing out certain granules, etc., in special objects a 
larger amount of alkali in the water is necessary. In this 
case add to 20 c.c. of the water, shortly before mixing with 
the staining fluid, an additional drop of alkali solution. 

S. B. Wolbach suggests, after considerable experience with 
the method, the following modifications : 

2. Clear in cedar oil instead of xylol. 

4. Use the weak solution of iodin in 70 per cent, alcohol. 



394 PATHOLOGICAL TECHNIQUE. 

6. The dilution should be 60 drops of the stain to 100 c.c. 
of distilled water, 10 c.c. of methyl alcohol, and 2 drops of a 
0.5 per cent, solution of sodium carbonate. The stain is re- 
placed twice by fresh mixtures during the first hour. 

7. Transfer the sections for differentiation directly from the 
staining mixture or, after a rapid passage through distilled 
water, into two changes of the following solution : 

Colophonium, 15 grams; 

Acetone, 100 c.c. 

The slides should be treated individually, and the differ- 
entiating fluid should be renewed as soon as the colopho- 
nium precipitated by the water fails to dissolve quickly. 
Differentiation takes place rapidly, fifteen to thirty seconds 
are usually sufficient. Should a deeper staining with blue be 
desired the amount of colophonium should be reduced. 

8. Pass the sections rapidly through — 

Acetone, 70 c.c. 

Xylol, 30 " 

followed by pure xylol and then by oil of cedar wood. 
Mount in oil of cedar wood. 

Rabies (Hydrophobia). — The diagnosis of this disease 
from a pathological standpoint is usually made by the pro- 
duction of experimental rabies in a rabbit by intradural 
inoculation with material from the nervous system of the 
animal suspected to have died of it. The poison of the dis- 
ease is found in the brain, spinal cord, salivary glands, and 
pancreas. For purposes of inoculation a piece (1 or 2 c.c.) 
of the medulla or brain, preferably the former, is rubbed up 
in a sterilized mortar with about 10 c.c. of sterilized distilled 
water. The resulting fluid is filtered through absorbent 
cotton, and then through filter-paper, to remove tissue- 
shreds. Of the clear fluid thus obtained 4 or 5 drops are 
injected beneath the dura of a rabbit by means of a hypo- 
dermic syringe, the skull being trephined with a small tre- 
phine about 4 mm. in diameter. The most favorable place 
for opening the skull is at a point in the median line just 
posterior to a line drawn through the middle of each eye. 



ANIMAL PARASITES. 395 

The symptoms of experimental rabies in the rabbit first 
manifest themselves after two weeks, never earlier, but they 
may not appear until later, not even until two months have 
passed. The first symptom is a weakness of the hind legs, 
followed by paralysis. The paretic condition soon extends 
to the fore legs, dyspnea appears, and death usually occurs 
in about three days after the onset of the symptoms. Para- 
lytic symptoms developing before two weeks are not due to 
infection with rabies, but to some other cause ; for instance, 
infection with the pneumococcus or other bacteria which 
may be present in the material inoculated. 

During the course of the disease the animal never appears 
stupid, with dull eyes, as in other infections, but remains 
" conscious," so to speak, until the last. 

It is claimed that the diagnosis of rabies may be made 
also by finding in the nerve-cells of the central nervous sys- 
tem peculiar bodies which are regarded as protozoa, and 
which are known as " Negri bodies," from the name of their 
discoverer. These bodies are generally round or oval, but 
may be irregular, pear-shaped, or triangular in form. They 
vary in diameter up to 23 jut. They contain small vacuoles, 
in some of which are granules of varying size and number; 
generally there is a central larger structure surrounded by 
smaller ones. In preparations stained by the eosin-methy- 
lene-blue method the bodies generally stain deeply with eosin, 
with the exception of the granules, some of which stain with 
the methylene-blue. 

Method of Demonstrating Negri Bodies (A. W. Williams 
and M. M. Lowden). — The bodies may be sought for in 
smear preparations or in sections. Pieces of gray brain- 
substance should be taken for examination from the cortex 
in the region of the fissure of Rolando (in the dog from 
around the crucial sulcus), from the hippocampus, and from 
the cerebellum. 

For demonstrating the bodies in sections the tissue should 
be fixed in Zenker's fluid, imbedded in paraffin, and stained 
by the eosin-methylene-blue method. 

For demonstrating the bodies in smear preparations the 



396 PATHOLOGICAL TECHNIQUE. 

following procedure is said to give the best results : A small 
bit of the gray substance of brain chosen for examination is 
cut out with a small, sharp pair of scissors and is placed 
about 1 inch from one end of a slide. The cut in the brain 
should be made at right angles to the surface and a thin slice 
taken, avoiding the white matter as much as possible. A 
cover-slip is now pressed down upon the piece of tissue until 




Fig. 125. — Ganglion-cells containing Negri bodies. 

it is spread out in a moderately thin layer, then the cover-slip 
is moved slowly and evenly over the slide to its other end. 
The preparation is then dried in the air and fixed in methyl- 
alcohol for about five minutes. It is then stained by Giemsa's 
method for malarial parasites (see page 391). It may also 
be fixed in Zenker's fluid, washed in alcohol, and stained by 
the eosin-methylene-blue method as a section affixed to the 

slide. . ... . 

Spirochetes of Relapsing Fever.— These spirilla, first 

discovered by Obermeier in 1873, occasionally are seen clinic- 



ANIMAL PARASITES. 397 

ally in this country. They are present in varying numbers 
in the circulating blood before and during the febrile par- 
oxysms to which the organism gives rise. 




'#• ^Rfl i, 



Fig. 126. — Spirochetes of relapsing fever. Smear preparation from blood; 
stained by Wright's blood-stain. X 1500. (Photo, by L. S. Brown.) 

Syphilis. — Methods of Demonstrating- Treponema Pal- 
lidum (Spirochete Pallida) in Smear Preparations. — The 
lesions are to be cleansed from any adherent exudate. The 
smear preparations are to be made from the juice of the tissue 
obtained by pressure and scraping. An excess of blood 
should be avoided. The preparations are then dried in the 
air and may be stained by the following methods : 

1. Method with Wrighfs Blood Stain. — Place in a test- 
tube 10 c.c. of distilled water, 1 c.c. of the blood-staining 
fluid, and 1 c.c. of a 0.1 per cent, solution of potassium carbo- 
nate. Heat to boiling and cover the preparation with the 
hot mixture. After three or four minutes, when the fluid on 
the preparation has become of a violet color and a thin 
yellow metallic scum has formed on the surface, pour off 
and again cover the preparation with the hot mixture after 
again heating in the tube. Repeat this once more. Dry 
and mount. 

The material should be thinly spread on a cover-glass, not 
on a slide, the cover-glass to be held level with forceps during 
the staining. 

The treponemata should stain intensely violet. 

2. Giemsds Method. — This is the same as his method for 
staining malarial parasites (see page 391), except that the 



39^ 



PA THOL O GICA L TE CHNIQ UE. 



preparation is fixed in absolute alcohol for fifteen to twenty 
minutes, and that to the water used for diluting the staining 
fluid I to io drops of a o.i per cent, solution of potassium 
carbonate is added. Preparations which are overstained may 
be differentiated by washing in distilled water for one to fif- 
teen minutes. 

Recently it has been shown that heating the diluted stain- 
ing fluid on the preparation stains the treponemata much 
more intensely. This modified method is as follows : 

Ten drops of Giemsa's staining fluid are mixed by gently 
shaking with io c.c. of distilled water immediately before 




\j> 



f 




Fig. 127. — Treponema pallidum in smear preparation (Goldhorn). 



proceeding to the staining. The preparation is fixed in abso- 
lute alcohol fifteen minutes, or by drawing three times 
through the flame. It is then covered with the diluted stain- 
ing fluid and warmed until a slight steam arises over the 
flame, and allowed to cool about fifteen seconds, when the 
diluted staining fluid is poured off and replaced by fresh 



ANIMAL PARASITES. 399 

fluid, and this again warmed to steaming and allowed to cool 
for about fifteen seconds. This process is repeated four or 
five times, after which the preparation is washed, dried, and 
mounted in balsam. In this modified method the staining 
of the parasites is intensely dark red. It is important that 
the slide or cover-glass be free from grease, and that the test- 
tube and the cover-glass or slide forceps be clean, free from 
acid, and from any precipitated stain. 

3. Beniari's Method. — Thoroughly mix on a cover-glass one 
or two loopsful of 2 per cent, aqueous solution of Congo red 
with a small amount of serum or exudate from the lesion. 




Fig. 128. — Treponema pallidum in a smear preparation stained by Benian's method; 
X 1500 (Wright and Brown). 

Spread the viscid mixture evenly and rather thinly and dry 
in the air; then wash with 1 per cent, aqueous hydrochloric 
acid, drain off at once, and again dry in the air. Do not 
wash in water or blot. 

The spirochetes appear white and unstained in a blue 
ground, which should be homogeneous. Too large a propor- 
tion of exudate results in an unsuitable granular ground. 

Serum or exudate which has been preserved by drying on 
glass is available for use by this method. 

4. Fontanas Method. — 1 . Dry the smear preparation in the 
air without heating. 



400 PATHOLOGICAL TECHNIQUE. 

2. Wash several times with a mixture consisting of I c.c. 
of glacial acetic acid, 20 c.c. of formalin, and 100 c.c. of 
distilled water. 

3. Wash in water and cover with a 1 per cent, aqueous 
carbolic acid solution in which 5 per cent, tannic acid has 
been dissolved. Heat until steam arises and allow to cool for 
thirty seconds. 

4. Wash in water, cover with the silver solution described 
below, heat until steam arises, and allow to cool for thirty 
seconds. 

5. Wash, dry, and mount. 

The spirochetes appear brown to black. 

The silver solution must be freshly prepared and is made 
by adding dilute ammonium hydrate solution drop by drop 
to a i per cent, solution of silver nitrate in distilled water 
until a faint turbidity appears. Excess of ammonium hy- 
drate must be avoided, for this clears up the turbidity, and 
such a solution is not suitable for use. 

5. Ghoreyeb's Method. — In this method the following solu- 
tions are used: 

1. One per cent, aqueous solution of osmic acid. 

2. Liquor plumbi subacetatis, diluted one hundred times 
with distilled water. This diluted solution should be freshly 
prepared. 

3. Ten per cent, aqueous solution of sodium sulphid. A 
thin smear is preferable. No heat fixation is necessary. 

The smear is stained as follows : 

1. Cover with osmic acid solution for thirty seconds. 

2. Wash in water. 

3. Cover with lead subacetate ten seconds. 

4. Wash in water. 

5. Cover with sodium sulphid solution ten seconds. 

6. Wash in water. 

This process is gone through with three times. Following 
this the osmic acid solution is applied for thirty seconds, and 
the specimen is then washed in water, dried, and mounted in 
balsam. A thorough washing in running water is essential 
after the application of each solution to prevent the forma- 
tion of excessive precipitates. 



ANIMAL PARASITES. 401 

The osmic acid, the first time applied, acts as a fixative and 
a mordant. The lead unites with the albumin to form lead 
albuminate, a compound insoluble in water. The sodium 
sulphid transforms the lead albuminate into lead sulphid, and 
causes the preparation to become stained brown. The osmic 
acid turns the brown color to black. The spirochetes, bac- 
teria, and cellular detritus are stained black. 

6. India Ink Method of 'Bum. — Approximately equal parts 
of the juice from the lesion and of fluid India ink are quickly 
mixed together on a slide with the aid of a platinum loop, 




Fig. 129. — Treponema pallidum; smear preparation from a cutaneous papule ; 
stained by Ghoreyeb's method; X 1500 (photo, by L. S. Brown). 

spread thinly, and allowed to dry thereon. When dry, the 
preparation is ready to be examined directly with the oil- 
immersion objective without covering it with balsam and a 
cover-glass. If the material contains many cellular elements 
or detritus, it will be necessary to dilute the ink with water. 
The preparation should have a brown color. The spiro- 
chetes and bacteria appear as unstained bodies in a brown to 
black background. 

Some specimens of fluid India ink are said to contain 
spirochete-like bodies, and therefore the ink used should be 
known to be free from such. 

7. LevaditVs Method for Staining Treponema Pallidum in 
Sections. — 1. Pieces of tissue about 1 mm. thick are placed 
in 10 per cent, formaldehyde for twenty -four hours. 

2. Rinse in water and place in 95 per cent, alcohol for 
twenty-four hours. 

3. Place in distilled water until the tissue sinks to the 
bottom of the container. 

26 



402 PATHOLOGICAL TECHNIQUE. 

4. Place in a 1.5 or 3 per cent, solution of nitrate of silver 
and keep in the incubator at 38 C. for three to five days. 
The stronger solution of nitrate of silver is preferable for 
tissues removed during life. 

5. Wash in distilled water and place in the following solu- 
tion for twenty-four to seventy-two hours at room tempera- 
ture : 

Pyrogallic acid, 2-4 gm. ; 

Formaldehyde, 5 c.c. ; 

Distilled water, 100 c.c. 

6. Wash in distilled water. 

7. Dehydrate in alcohol, clear in chloroform, and embed 
in paraffin in the usual manner. 

The treponemata are stained intensely black by the pre- 
cipitation of metallic silver upon them. The reticulum stains 
brown, while the other elements of the tissue generally are 
of a yellow color. The sections may be counterstained with 
some aniline dye, but this is of doubtful advantage. 

8. Eyenes and Sternberg's Method for Staining Treponema 
Pallidum in Sections. — 1. Fix well in 10 per cent, formalin. 
Cut sections, by freezing or in celloidin, as thin as possible. 

2. Wash the sections in distilled water, and place them in 
a 1 per cent, aqueous solution of silver nitrate for thirty to 
thirty-five minutes in the incubator or longer at room tem- 
perature in the dark. The period of time in the incubator is 
important. 

3. Place the sections in the following mixture until they are 
dark brown (one to two minutes), removing them before a 
precipitate occurs : 

2.5 per cent, aqueous solution of siver nitrate, 10 c.c. ; 
10 per cent, aqueous solution of gelatin (warm), 10 " 
50 per cent, aqueous solution of gum arabic, 10 " 
5 per cent, aqueous solution of hydroquinone, 5 " 

The ingredients are to be well mixed both before and after 



ANIMAL PARASITES. 



403 



the addition of the hydroquinone, which should be added 
last. 

4. Wash for one to two minutes in 10 per cent, aqueous 
solution of sodium hyposulphite. 

5. Wash in water, dehydrate, clear, and mount. 

The microscopical appearances are essentially the same as 
those produced by Levaditi's method. 





A B c 

Fig. 130. — Taenia mediocanellata : A, head darkly pigmented; B, ripe joint, 
X 6 ; C, egg of taenia mediocanellata. 

Tape-worms. — It is not always easy to recognize the 
kind of tape-worm by a single segment passed with the feces, 




^ 
fi 



Fig. 131. — Echinococcus : scolices, hooks (Heller). 



because the uterus, which furnishes the most characteristic 
points of difference, is not developed in the young segments 



4 04 



PA THOL O GICAL TECHNIQ UE. 



and is atrophied in the old ones. When the whole worm is 
obtained the problem is much simpler. The uterus is best 




A 
Fig. 1.32. — Dibothriocephalus latus : A, head; B, ripe joint, X6; C, egg of 
dibothriocephalus latus (Heller) ; D, egg with developed embryo (Leuckart). 

made out by squeezing a segment between two slides and 
holding it up to the light. The heads are examined under 
the microscope in water, salt solution, or glycerin. 




Fig. 133. — Segments of taenia 
saginata (after Stein). 



Fig. 134. — Segments • Fig. 135. — Segments 
of bothriocephalus latus of taenia solium (after 
(after Stein). Stein). 



Taenia Solium (Fig. 144). — Head has four suckers and a 
circle of hooklets; uterus is noticeably but little branched. 
The genital tract opens laterally. The eggs develop into the 



1 



ANIMAL PARASITES. 



405 



The genital tract opens laterally. The eggs develop into the 
cysticerci cellulosse, which are not infrequently found in 
man. The scolex is obtained for examination by tearing 
open the cyst and examining the inner wall. The suckers 
and hooklets are best studied after mounting fresh and 
pressing under a cover-glass. 

Taenia Mediocanellata s. Saginata (Fig. 130). — Head 
has four strong suckers, but no hooklets; uterus is very 





Fig. 136. — Comparative size of eggs of intestinal parasites : a, taenia solium ; 
b, taenia mediocanellata; c, ascaris lumbricoides ; d, trichocephalus dispar; e, 
oxyuris vermicularis (after Strumpell). 

much branched, segments show marked muscular develop- 
ment. The genital tract opens laterally. The eggs develop 
into cysticerci, which do not occur in man. 

Taenia Echinococcus (Fig. 131) occurs in dogs. The 
echinococcus cysts which occur in man are recognized by 
the very characteristic laminated structure of the cyst-wall. 
The heads of the scolices have four suckers and a double 
circle of hooklets. 

Dibothriocephalus latus (Fig. 132). — The opening of the 
genital tract lies in the median line. The head is flattened, 
and has two small suckers situated at the sides. 

Schistosoma Haematobium (Distomum Haema- 
tobium, Bilhar^ia). — The male and female parasites occur 
in the branches of the portal system, especially in the veins 
of the bladder and rectum, and in the liver. The ova escape 
from the blood-vessels into the bladder and occasion violent 
inflammation. The process may extend to the kidneys. The 
ova also infect the rectum, causing a sort of dysentery, and 
may involve even the appendix. The ova, with their pointed 



406 PATHOLOGICAL TECHNIQUE. 

spines, are characteristic, and may be found by microscopical 
examination in the urine and feces. The spines are usually 
situated at one end, but may occur anywhere in the periphery. 




Fig. 137. — Schistosoma haematobium. Ovum from fasces showing pointed 
spine on one side ; X 300 (photo, by L. S. Brown). 




Fig. 138. — Schistosoma haematobium. Ovum from urine showing terminal 
spine ; X 375 (photo, by L. S. Brown). 

Round-worms. — The embryos of the filaria sanguinis 
hominis or filaria Baiter of ti (Fig. 139) are examined for in 
suspected cases by mounting a drop of the fresh blood or 



ANIMAL PARASITES. 



407 



of the chylous or bloody urine on the slide and examining 
under low power. They are readily detected when present 
on account of their very active movements. Six species 




Fig. 139. — Adult male filaria Bancrofti ; X 10 (after Lothrop and Pratt). 

have been described, but the filaria nocturna is the only 
one that is known to be pathogenic. The blood should 




Fig. 140. — Photomicrographs of living filariae sanguinis hominis ; X 250, a, trom 
hydrocele fluid; b, from blood (after Lothrop and Pratt). 

be examined during the resting hours of the patient, as 
at night for day-workers and during the day for night- 
workers. Permanent specimens can be made by fixing 



408 PATHOLOGICAL TECHNIQUE. 

ordinary cover-slip preparations of the blood or chylous 
fluid by heat or by the use of a saturated solution of corro- 
sive sublimate, and staining for a few seconds with Loffler's 
methylene-blue or with a 2 per cent, aqueous solution of 
thionin. 

In the case of suspected hookworm disease the feces are to 
be examined microscopically for the eggs of the parasite, or 




Fig. 141. — Two eggs of the hookworm in feces, each containing several embry- 
onic cells; X 375. (On C. L. Overlander.) 

for the adult worms after the administration of an anthelmin- 
tic. The finding of the eggs may be facilitated by mixing 
the feces with a nine-tenths saturated aqueous solution of 
sodium chloride in a test tube and examining microscopically 
the superficial portions of the fluid, where any eggs present 
will accumulate because their specific gravity is less than 
that of the fluid. This procedure was first recommended by 
Bass. In fresh feces, eggs containing several embryonic 
cells are most common. (See Fig. 141.) 

Trichinellse (Figs. 142, 143) are obtained from the fresh 
muscle by means of teasing. A quick method is to squeeze 
small bits of tissue between two slides and examine with a 
low power. Pieces of muscle nearest the insertion of the 
tendon are chosen from the diaphragm or from the muscles 
of the jaws. Encapsulated and calcified trichinellae are cleared 
up by means of acids. 



ANIMAL PARASITES, 



409 



In hardened tissues the trichinellae are best studied in 
longitudinal sections of the muscle-fibers. 

The other round-worms which sometimes occur in the 





Fig. 142. — Living embryos 
(Heller). 



Fig. 143. 



- Encapsulated trichina 
(Leuckart). 



intestinal tract can be recognized with the naked eye. Their 
eggs must be looked for with the microscope. 

The embryo trichinellae may be demonstrated in the blood 
by withdrawing some blood with a syringe from a vein in the 






Fig. 144. — Taenia solium : A, head enlarged ; B, ripe joint, X 6 ; C, egg of 
taenia solium (Heller). 

arm, washing it with 3 per cent, acetic acid, centrifugalizing, 
and examining the sediment (W. H. Herrick and T. C. 
Janeway). 



CLINICAL PATHOLOGY. 



EXAMINATION OF THE BLOOD. 

The blood is conveniently obtained from the lobe of the 
ear by puncture with a Hagedorn needle. The part should 
be previously cleaned with alcohol and thoroughly dried. 
The free border of the lobe "of the ear is preferable because it 
is convex, which is of advantage in making cover-glass prep- 
arations from small drops of blood. 

Method of Counting the Red and White Blood- 
corpuscles. — The Thoma-Zeiss hemocytometer, or blood- 
counting apparatus, is generally employed, and consists of a 
glass slide, on which the blood-corpuscles are counted, and 
two graduated pipettes for mixing the blood and the diluting 
fluid. The counting slide has a square plate of glass cemented 
on its surface, and a circular opening in the center of this 
plate is nearly filled by a glass disc y 1 ^- mm. thinner than the 
square plate which surrounds it. A series of horizontal and 
vertical lines on the surface of the disc divides it into squares, 
the sides of which are -£$ mm. long. Additional lines placed 
close together divide this surface into quadrants. Each 
quadrant contains sixteen of the small squares. 

Each pipette consists of a capillary tube, which extends 
into an ovoid chamber above and is provided with a short 
piece of rubber tubing and a bone mouth-piece. The cham- 
ber contains a glass pearl which assists in mixing the blood 
and diluting fluid. For counting the red blood-corpuscles the 
blood is diluted I : ioo or I : 200 by means of the pipette 
which has the figures 101 over the line above the ovoid 
chamber. The diluting fluid recommended is that of Gow- 
ers, the formula of which is as follows : 

410 



I 



CLINICAL PATHOLOGY. 
B 



411 










cV 




r\ 








- 


< 


p 


0^ 







^ 








Q 


^ 


^C 


~^J 


o° 


■' 




VJ 






c 





u 




-. 


O 


^9 


c=> 












| 




) O 




> 


O 













^ c 


O 

c 


0* 

C ^ 


) 

53 




c 









c 


O 


O 

O 

O 


O 

< 







a | 


£> 




r 


, O 








cS 









~-^ 









o> 




/^-\ 
^ 





















ay 






a 



Fig. 145. — Thoma-Zeiss blood-counting apparatus (Limbeck) : A, melangeur ; 
a, capillary tube in which the blood is taken ; b, chamber for mixing the blood 
with the diluting solution ; c, glass ball to aid in mixing the blood with the dilut- 
ing solution ; B, cross-section of the chamber in which the blood is counted ; 

C, section of the field on which the blood is counted, showing thirty-six squares ; 

D, diagram of the whole field. 



4 J 2 PATHOLOGICAL TECHNIQUE. 

Sodii sulphat, gr. 112; 

Acid, acet., 3v ; 

Aquae, 3iv. 

With the mouth-piece of the rubber tube in the mouth, the 
blood is sucked up to the mark 0.5 or 1.0 on the capillary tube 
of the pipette, and then the tip of the tongue is placed firmly 
over the hole in the mouth-piece. This prevents the blood- 
column from sinking or air from entering below while the tip of 
the pipette is being wiped and immersed in the diluting fluid. 
This part of the test requires the utmost precision and avoid- 
ance of delay. It is necessary, therefore, to keep the eyes 
constantly fixed on the cariillary tube in order to note any 
variation in the blood-column. Rapidly wipe the tip of the 
pipette to remove the blood from the outside, and then im- 
merse the tip in the diluting fluid. Suck the fluid up to the 
mark 101, close the ends of the pipette with the thumb and 
middle finger, and shake the pipette for two minutes. If 
the ends of the pipette are not completely closed during this 
process, some of the fluid will escape. At the end of 
two minutes allow two drops to escape from the pipette 
before examination, because the fluid in the capillary tube is 
unmixed with blood. Then allow a drop to escape upon the 
central part of the counting slide. This drop should com- 
pletely fill the depression after the cover-glass has been 
applied. A little practice is necessary in order to estimate 
the size of the drop required. A moderately thick cover- 
glass should be slid over or carefully laid upon the square 
raised surface, and pressure applied to the edges until the 
Newton color-zone can be seen between the cover-glass and 
the square raised surface beneath. Never press on the 
center of the cover-glass. Allow the blood-corpuscles to 
settle a minute or two before counting. 

The corpuscles are estimated as follows : One side of a 
small square is -£$ mm. long ; the enclosed square surface is 
4^- mm. The distance between the cover-glass and the 
disc is .1 mm. which gives a cubic capacity of 4^00" c - mm - 
for each square. To estimate the number of corpuscles in 



CLINICAL PATHOLOGY. 413 

I c.mm. of blood, multiply the number of corpuscles counted 
by 4000, and then by the number representing the amount 
of dilution, 100 or 200 as the case may be, and divide the 
result by the number of squares counted. 

Corp. X dilution X 4000 1 

c ■ - — corpuscles in 1 c.mm. 

Squares counted 

To avoid counting any of the corpuscles twice, always 
begin at the upper left-hand square of a quadrant and count 
four squares downward. Count all the corpuscles which 
touch the upper and left-hand lines of a square, together 
with the corpuscles in the square. Never count the cor- 
puscles touching the right-hand or lower double lines of a 
quadrant. In order to make an accurate count it is neces- 
sary to count at least 1200 red corpuscles. 

If air-bubbles are present when the cover-glass is applied, 
it is necessary to clean the slide and use a fresh drop of the 
diluted blood. Before beginning the count examine the 
various quadrants with a low-power objective, to see if the 
corpuscles are evenly distributed. If they are not, it will be 
because the blood is not thoroughly mixed, and the slide 
should be washed and the pipette well shaken. Before ex- 
amining a second drop of the diluted blood shake the 
pipette for two minutes as before. The results of three drops 
should be averaged. 

For counting the white blood-corpitscles the pipette, gradu- 
ated so as to give a dilution of 1 to 10 or 1 to 20, is employed. 
The white corpuscles are estimated in the same way as the 
red corpuscles, except that the dilution 10 or 20 is substituted 
for 100 or 200. This necessitates a fresh drop of blood. For 
a diluting fluid for counting the white corpuscles a \ or \ 
per cent, solution of acetic acid may be used. This solution 
destroys the red corpuscles. 

After use the pipette should be cleaned with water followed 
by alcohol, and finally with ether until it is dry inside. Any 
coagulated albumen on the inside of the pipette may be re- 
moved by filling the pipette with the following solution and 
keeping it in the incubator for some hours : 



4H PATHOLOGICAL TECHNIQUE. 

5 gms. carbonate of sodium, 
\ gm. pancreatin, 
IOOO ex. water, 

A few drops of chloroform. 

In cleaning the counting slide water should be used. If 
it is necessary to employ alcohol for any reason, it must be 
used rapidly and the slide washed with water, because alcohol 
dissolves the cement by means of which the glass plates are 
attached to the slide. 

Wright's Method of Counting the Blood-platelets. 
— The blood is mixed with a diluting fluid, in the proportion 
of I to ioo, by means of the pipette used for counting red 
blood-corpuscles, and the counting is done in the ordinary 
blood-counting chamber with a high-power dry objective. 
In order to render the platelets more clearly visible, the 
specially thin cover-glass of Zeiss with central excavation is 
used (cover-glass No. 146, Zeiss catalogue). This may be 
obtained from Eimer & Amend, 205 Third Avenue, New 
York. The diluting fluid consists of three parts of an 
aqueous solution of potassium cyanide (1 : 1400) and two 
parts of an aqueous solution of brilliant cresyl-blue (1 : 300). 
These two solutions must be kept in separate bottles, and 
mixed and filtered immediately before using. Of course the 
pipette should be well shaken before withdrawing the sample 
for counting. After the counting chamber is filled it is left 
at rest for ten or fifteen minutes, in order that the blood- 
platelets may all settle to the bottom of the chamber and be 
more easily and accurately counted. 

The platelets appear as sharply outlined, round or oval or 
elongated, lilac-colored bodies, some of which form a part 
of a small sphere or globule of hyaline unstained substance. 
The red cells are decolorized and appear only as " shadows," 
so that they do not obscure the platelets. The nuclei of the 
white cells are stained a dark blue, the protoplasm light 
blue. If the technique is correct, there should be no precipi- 
tate in the preparation. 

The cresyl-blue solution is permanent, but should be kept 
on ice in order to prevent the growth of yeasts. The cyanide 



CLINICAL PATHOLOGY. 415 

solution should be made up at least every ten days. It is, 
of course, necessary that the solution be made from pure 
potassium cyanide which has not undergone decomposition. 
As already stated, the two solutions must be mixed and fil- 
tered immediately before using, because after filtration, if the 
mixture is allowed to remain exposed to the air for a short 
time, a precipitate will form in it. After the diluting fluid 
has been mixed with the blood in the pipette, however, no 
precipitate forms and, as the platelets do not quickly break 
up in the mixture, the counting may be done after some 
hours, if necessary. For example, a count immediately after 
filling the pipette was 258,000, and another count from the 
same filling of the pipette, made eighteen hours later, was 
253,000. 

A proper technique yields a remarkably even distribution 
of the platelets in the chamber. For all practical purposes 
the counting of the platelets in 100 small squares is sufficient, 
but for greater accuracy all 400 small squares should be 
counted, or 200 small squares in each of two fillings of the 
chamber. 

The following is a sample count to show the even distri- 
bution of the platelets in the counting chamber : 

Platelets in Chamber No. i. Chamber No. 2. 

20 small squares 22 21 21 25 

" 27 28 27 23 

" 27 24 27 22 

" 23 30 22 25 

" -_2I _2I 23 31 

I20 124 I20 126 

By this method the platelet count of normal adults is found 
to vary from 226,000 to 367,000 per cubic millimeter, the 
general average being 297,000. 

Cover-glass Preparations. — The blood must be spread 
extremely and uniformly thin. If this is done, the blood 
dries very quickly, and the red blood-corpuscles retain their 
shape and are not crowded together and lying over one an- 
other. To obtain such a result it is essential that the cover- 
glasses should be absolutely clean ; that there should be no 
delay in bringing the cover-glass which has the drop of 



416 PATHOLOGICAL TECHNIQUE. 

blood on its surface in contact with a second cover-glass; 
and that the drop of blood should be quite small. The fol- 
lowing method gives the best results : The procedure is ren- 
dered much easier if some one is present to assist. This 
person places a finger beneath the lobe of the ear in order 
to raise it slightly without pressing upon it, and with a clean 
compress wipes away the blood as fast as it flows with a 
quick motion of the hand. This is done to prevent coagu- 
lation, which occurs very quickly, and prevents the drop of 
blood from spreading between the cover-glasses. It takes a 
little time for the blood to' spread, the cover-glasses to be 
separated and laid down, and fresh ones picked up ; and if, 
during this time, some one wipes away the blood as fast as 
it flows, much better preparations are obtained. If, in spite 
of this, as often happens, the blood coagulates about the 
opening, one end of the compress can be slightly moistened 
with water and passed over the opening and the surface dried 
quickly. The blood then flows freely again. A drop of 
blood a little larger than a pin-head is sufficient. Grasp the 
edge of the cover-glass with a pair of spring forceps, pick up 
a second cover-glass with a pair of plain forceps. Both 
pairs of forceps must be especially prepared by having the 
inner surfaces of the points ground smooth. The cover-glass 
in the spring forceps is held horizontally just below the ear, 
and the other cover-glass, held with the other forceps, is 
touched lightly on the blood and immediately dropped on 
the first one. If the cover-glasses are dry and clean and the 
blood has not begun to coagulate, it spreads at once in a 
thin film between the glasses. The glasses are then drawn 
apart with a rapid sliding motion by means of the forceps, 
waved in the air a few seconds, and laid down with the 
blood-surface uppermost. The layer of blood cannot be too 
thin, but it can easily be too thick. The cover-glasses 
should never be pressed together to make the blood spread. 
Considerable practice is required before one becomes pro- 
ficient. The specimens may be fixed by heat in a thermostat 
at a temperature between I io° and 120 C. (Ehrlich's 
method). This is objectionable on account of the time and 
apparatus required. A practical modification of this method 



CLINICAL PATHOLOGY. 417 

is to heat the cover-glasses on a brass plate for an hour at a 
point on the plate where water boils. The plate should be 
about \ of an inch thick and from 15 to 18 inches long. It 
should be heated from one end to a constant temperature. 
Test the degree of heat with drops of water and select a 
part where the water boils. At a point nearer the flame it 
will be found that the water sputters and rolls about, indi- 
cating too high a temperature. After putting the cover- 
glasses, with the blood-side uppermost, upon the selected 
point, it is necessary to test the degree of heat from time to 
time, and perhaps to shift the cover-glasses. 

Smear preparations on slides are easier to prepare than 
those on cover-glasses, and in many ways they are more 
satisfactory. All the precautions as to cleanliness described 
for cover-glasses are to be taken. The smear is made by 
placing a small drop of blood upon the slide near one end 
and immediately spreading the blood by drawing the edge 
of the end of another slide through the drop and along the 
surface of the slide to its other extremity. 

Methods of Staining:. — Of the many staining fluids 
which have been employed to differentiate the white cor- 
puscles, it is necessary to mention only two, which have 
been found to answer all purposes. 

Ehrlich's Triple Stain. — The formula is as follows : 

Orange G, saturated aqueous solu- 
tion, 120 to 135 c.c. ; 
Distilled water, 100 " 



Acid fuchsin, saturated aqueous 




solution, 


65 "■ 


Distilled water, 


100 " 


Absolute alcohol, 


100 " 


Methyl green, saturated aqueous 




solution, 


125 c.c. 


Distilled water, 


100 " 



Absolute alcohol, 100 

Glycerin, 100 

27 



418 PATHOLOGICAL TECHNIQUE. 

The various ingredients are prepared separately as indi- 
cated by the dotted lines, and are afterward mixed gradually. 
The mixture must stand for several weeks before using. It 
is advisable to withdraw by means of a pipette some of trie 
staining fluid from the middle portion without disturbing the 
bottom. 

The cover-glass preparations should be stained from six 
to eight minutes, washed thoroughly with water, dried, and 
mounted in Canada balsam. The neutrophilic granules are 
stained violet ; the eosinophilic, a bright red; the nuclei of 
the neutrophilic and the eosinophilic cells are a greenish- 
blue ; the nuclei of the lymphocytes, a deep blue ; the 
nuclei of the large mononuclear cells, a pale blue ; the red 
corpuscles, copper color; and the nuclei of the red cor- 
puscles, if any be present, a more intense blue than the 
nuclei of the lymphocytes. For some unexplained reason 
this stain is not always uniform in its action. 

It is sometimes difficult to distinguish a nucleated red cor- 
puscle from a lymphocyte. It is well to remember, there- 
fore, that the nuclei of red corpuscles stain more intensely 
than other nuclei, and have very sharply defined outlines, 
and by careful focusing it is seen that the surrounding 
stroma is stained the same color as the other red corpuscles. 

'Wright's Stain. — This staining fluid is an improvement 
on one devised by W. B. Leishman, because it requires only 
a few hours and an ordinary steam sterilizer for its prepara- 
tion, while Leischman's required at least eleven days and 
the employment of a thermostat regulated at 65 ° C. Leish- 
man deserves great credit for originating a method of stain- 
ing blood-films and malarial parasites which combines the 
important " Romanowsky " staining with the great advan- 
tages of the methyl-alcohol method of Jenner. Wright's 
stain is applied in the same manner and gives the same 
results. 

It is preferred to Ehrlich's stain, because it does not 
require the difficult and uncertain fixation of the blood-film 
by heat and because it gives constantly satisfactory results 
even in the hands of inexperienced workers. 



CLINICAL PATHOLOGY. 419 

This stain makes visible in the blood smear not only all 
that the Ehrlich's stain does, but more, for it gives the dif- 
ferential Romanowsky staining to mast-cells, blood-plates, 
certain degenerate products in the red corpuscles, 'and to 
malarial and other protozoan parasites, thus accomplishing 
at one and the same time all that which usually requires the 
employment of several special staining methods separately 
applied. 

It is prepared as follows : 

To a 0.5 per cent, aqueous solution of sodium bicarbonate 
add methylene-blue (B.X. or " medicinally pure ") in the 
proportion of 1 gm. of the dye to each 100 c.c. of the so- 
lution. Heat the mixture in a steam sterilizer at ioo° C.for 
one full hour, counting the time after the sterilizer has be- 
come thoroughly heated. The mixture is to be contained in 
a flask, or flasks, of such size and shape that it forms a layer 
not more than 6 cm. deep. After heating, the mixture is 
allowed to cool, placing the flask in cold water if desired, 
and is then filtered to remove the precipitate which has 
formed in it. It should, when cold, have a deep purple red 
color when viewed in a thin layer by transmitted yellowish 
artificial light. It does not show this color while it is warm. 

To each 100 c.c. of the filtered mixture add 500 c.c. of a 
O.I per cent, aqueous solution of " yellowish, water-soluble " 
eosin and mix thoroughly. Collect on a filter the abundant 
precipitate which immediately appears. When the precipitate 
is dry, dissolve it in methylic alcohol (Merck's " reagent ") in 
the proportion of 0.1 gm. to 60 c.c. of the alcohol. In 
order to facilitate solution the precipitate is to be rubbed up 
with alcohol in a porcelain dish or mortar with a spatula or 
pestle. 

This alcoholic solution of the precipitate is the staining 
fluid. It should be kept in a well-stoppered bottle because 
of the volatility of the alcohol. If it becomes too concen- 
trated by evaporation and thus stains too deeply, or forms a 
precipitate on the blood smear, the addition of a suitable . 
quantity of methylic alcohol will quickly correct such faults. 
It does not undergo any other spontaneous change than that 
of concentration by evaporation. 



420 PATHOLOGICAL TECHNIQUE. 

A most important fault met with in the working of some 
samples of this fluid is that it fails to stain the red blood- 
corpuscles a yellow or orange color, but stains them a blue 
color which cannot readily be removed by washing with 
water. This fault is due to a defect in the specimen of eosin 
employed. It can be eliminated by using a proper " yellow- 
ish, water-soluble " eosin. 

Method of Staining Blood Films. — -The films of blood which 
should be spread thinly are allowed to dry in the air. 

1. Cover the film with a noted quantity of the staining 
fluid by means of a medicine-dropper. 

2. After one minute add to the staining fluid the same 
quantity of distilled water by means of the medicine-dropper, 
and allow it to remain for two or three minutes, according to 
the intensity of the staining desired. A longer period of 
staining may produce a precipitate. Eosinophilic granules 
are best brought out by a shorter period of staining. The 
quantity of diluted fluid on the preparation should not be so 
large that some of it runs off. 

3. Wash the preparation in water for thirty seconds, or 
until the thinner portions of the preparation become yellow 
or pink in color. 

4. Dry and mount in balsam. 

Films more than a few hours old do not stain as well as 
fresh ones. 

Microscopical Appearances in Blood-films Stained "with 
Wright's Stain. — The red cells are orange or pink in color. 
Polychromatophilia and punctate basophilia or granular 
degeneration are well brought out. The nucleated red cells 
have deep-blue nuclei, and the cytoplasm is usually of a 
bluish tint. 

The lymphocytes have dark purplish-blue nuclei and 
robin's-egg-blue cytoplasm in which a few dark-blue or 
purplish granules are sometimes present. 

The polynuclear neutrophilic leucocytes have a dark-blue or 
dark lilac-colored nucleus, and the granules are usually of 
a reddish-lilac color. 

The eosinophilic leucocytes have blue or dark lilac-colored 



CLINICAL PATHOLOGY. 42 1 

nuclei. The granules have the color of eosin, while the 
cytoplasm in which they are imbedded has a blue color. 

The large mononuclear leucocytes appear in at least two 
forms. Each form has a blue or dark lilac-colored nucleus. 
The cytoplasm of one form is pale blue and of the other 
form is blue with dark-lilac or deep-purple-colored granules, 
which are usually not so numerous as are the granules in 
the polynuclear neutrophilic leucocytes. 

The mast-cells appear as cells of about the size of poly- 
nuclear leucocytes with purplish or dark-blue stained, 
irregular-shaped nuclei, and with cytoplasm, sometimes 
bluish, in which numerous coarse spherical granules of 
variable size are embedded. These granules are of a dark 
purple color and may appear almost black. 

The myelocytes have dark-blue or dark lilac-colored nuclei 
and blue cytoplasm in which numerous dark-lilac or red- 
dish-lilac-colored granules are imbedded. In leukemia more 
color differences are brought out among the leucocytes than 
by the ordinary methods of staining. 

The blood-platelets are well stained. In the best prepara- 
tions they generally appear as round or oval blue bodies 
with smooth or finely irregular margins, containing, chiefly 
in their central portions, many small violet to purplish gran- 
ules. They are usually of a diameter of one-third to one- 
half that of the red blood-corpuscles. They frequently occur 
in groups and masses. Occasionally elongated forms are seen 
which may have a length of six or more times their width. 
These are, according to Wright's observations, the detached 
larger pseudopods of the giant cells of the bone-marrow, 
just as the smaller platelets are detached smaller pseudopods 
or fragments of the larger pseudopods of the same cells. 

Method of Examining Blood without Drying or 
Fixation. — A small quantity of a 1 per cent, aqueous solu- 
tion of brilliant cresyl-blue is dried upon a slide in such a 
manner that a thin, transparent film of the dried dye is ob- 
tained. Upon this a small drop of blood is placed and imme- 
diately covered with a cover-glass. The blood is spread out 
in as thin a layer as possible under the cover-glass, using 



422 PATHOLOGICAL TECHNIQUE. 

gentle pressure if necessary. The plasma immediately dis- 
solves the dye on the surface of the slide, and in the course 
of some minutes the following appearances will be seen 
upon examination with an oil immersion objective : the 
white blood-corpuscles are stained blue and show all details 
of their structure; the blood-platelets remain intact and 
appear as irregular-shaped hyaline bodies, each with a round, 
blue-staining, somewhat granular central structure. In the 
interior of a few of the red blood-corpuscles a blue-staining 
reticulum of delicate, contorted filaments will be seen. In 
anemic blood many more corpuscles contain this reticulum. 
The blood-corpuscles do not become abnormal in shape, but 
retain their natural cup shape. Fibrin does not form. 

The preparation may be kept under observation for hours 
before marked disintegrative changes occur in the formed 
elements. Instead of the dye mentioned on page 421, 
Wright's blood-stain may be used. 

Sehult^e's Oxydase Reaction. — Many cells possess 
an oxydizing ferment, which they disclose by forming syn- 
thetically naphthol blue when they are treated first with 
a-naphthol and then with dimethyl-p-phenylendiamin. The 
method is particularly useful for differentating myelocytes 
from cells of the lymphocyte series ; the myelocytes give a 
positive reaction, while the lymphocytes are negative. 

The reaction is carried out as follows (two solutions are 
required) : 

1. One gram of a-naphthol is heated to boiling in 100 c.c. 
of distilled water until it melts and floats in the water. Pure 
potassium hydrate (about 1 c.c.) is added until all the naph- 
thol is dissolved. The solution appears at first yellow, later 
yellowish brown. 

2. One per cent, aqueous solution of dimethyl-p-phenylen- 
diamin (Merck), made at room temperature and filtered. 

Cover-glass preparations fixed in formaldehyde • vapor or 
frozen sections of formaldehyde fixed tissue are placed in so- 
lution (1) for a few minutes, and then in solution (2) for a 
similar length of time ; they should be moved gently back 
and forth in the solutions. 



CLINICAL PATHOLOGY. 423 

The preparations after being washed in distilled water 
should be mounted and examined in water or glycerin jelly. 
The granules, which exhibit the oxydase reaction, are stained 
deep blue. The preparations are not permanent. 

Graham's Alphanaphthol-pyronin Stain for the Oxy- 
dase Granules. 1 — A. Method for Blood Smears. — 1. Allow 
the smear to dry thoroughly in the air, then fix for one or 
two minutes in a freshly prepared mixture of 9 parts of 
95 per cent, alcohol and 1 part of strong formaldehyde 
solution. 

2. Wash in water and flood with the following alphanaph- 
thol solution: 

Alphanaphthol (Merck's "Recrystallized" 

or Merck's ' 'Reagent"), 1 gm. ; 

Forty per cent, alcohol, 100 c.c; 

Hydrogen peroxide, 0.2 " 

Allow a reaction time of four to five minutes. 

3. Wash and place in a dish of running water for about 
fifteen minutes. 

4. Stain two minutes with the following solution: 

Pyronin, 0.1 gm.; 

Aniline, 4.0 c.c; 

Forty per cent, alcohol, 96.0 

Dissolve the pyronin in the alcohol and add the aniline. 
The solution keeps well. 

5. Wash in water. 

6. Stain one-half to one minute with a 0.5 per cent, 
aqueous solution of methylene-blue (Grubler's B. X.). 

7. Wash in water, blot, and dry. 

8. Mount in neutral balsam. 

The resulting picture is much like that afforded by the 
Romanowsky stains, except for the greater prominence of 
the granules. The neutrophilic granules are usually very 
abundant, so that the cytoplasmic substance of the cell 
appears almost completely filled with them. They are some- 

1 Graham, G. S., "The Oxidizing Ferment of the Myelocyte Series of Cells 
and Its Demonstration by an Alphanaphthol-pyronin Method," Jour. Med. 
Research, 1916, xxxv, 231-242. 



424 PATHOLOGICAL TECHNIQUE. 

what irregular in form and size and are purplish-red in color. 
Occasional cells show fewer and more faintly stained granules. 
They may represent old degenerating forms of the cell. 
The eosinophilic granules are larger, somewhat lighter and 
more refractile, and have the appearance of spherical bodies 
with lighter staining centers. The mast-cell granule takes 
a more basic stain, so that it appears of a deep purple color. 
Myelocytes have granules v of varying number and size. 
Erythrocytes are greenish-yellow to pink; platelets, blue; 
nuclei of all cells, blue; cytoplasm, light blue. 

The best results are secured with fresh smears or with such 
as are not over a few days old. After ten days to two weeks 
the reacting substances begin to disappear, so that in older 
specimens many of the granules fail to stain and some cells 
may appear entirely devoid of them. The eosinophilic 
granule retains its staining power much longer than the 
neutrophilic. 

B. Method for Tissues. — The material must be formalin- 
fixed and freshly cut frozen sections must be used. After 
standing twenty-four to forty-eight hours in water the 
granules may fail to react. 

1. Stain rather lightly in alum-hematoxylin. The solu- 
tion must not be too acid. 

2. Wash in water, then for about five minutes in a saturated 
aqueous solution of lithium carbonate, and return to water 
for a few minutes. 

3. Stain ten minutes in a mixture made up by adding a 
2 per cent, aqueous solution of pyronin to the alcoholic 
alphanaphthol solution given above in the proportion of I 
drop of the pyronin to 2 c.c. of the alphanaphthol solution. 
The mixture must be prepared immediately before use. 
Evaporation should be prevented by staining in a closed 
container, such as a covered Stender dish. Shake gently 
from time to time to ensure even exposure. 

4. Wash in water, then place for fifteen to twenty minutes 
in a saturated aqueous solution of lithium carbonate. W 7 ash 
thoroughly in several changes of water. 

5. Differentiate and dehydrate in 80 per cent., followed by 
95 per cent., alcohol, transfer to a slide, and clear with xylol 
by the blotting method. 



CLINICAL PATHOLOGY. 42$ 

6. Mount in neutral balsam. 

The preparations show an intense red coloration of the 
granules. Nuclei are greenish-blue to blue. 

Cultures from the Blood during I^ife.— This pro- 
cedure is coming more and more into use for diagnostic 
purposes in cases of suspected bacterial infection of the 
circulating blood. The blood is obtained from one of the 
large veins at the flexure of the elbow by means of an anti- 
toxin or similar syringe, and in most cases is immediately 
mixed with an excess of sterile bouillon. About 10 c.c. or 
more of blood should be taken. A convenient way is to 
distribute the blood directly by means of the syringe in 
quantities of 3 or 5 c.c. among flasks, each containing 200 
to 400 c.c. of sterile bouillon. The blood is thus highly 
diluted in order to obviate its bactericidal action. 

The addition of sterile ascitic fluid to the bouillon in the 
proportion of I part to 3 or 4 parts of bouillon is desirable in 
cases in which streptococcus or pneumococcus is suspected. 

In the case of general infection with anaerobic bacteria 
the blood should be mixed immediately with melted agar- 
agar at 40 C. in test-tubes, in the proportion of 1 part to 
2 or 3 parts of agar-agar. The mixture should fill the 
culture -tubes to a height of about 8 cm. For the method 
of cultivating gonococci from the blood see page 273. 

The strictest aseptic precautions must be observed in ob- 
taining the blood and in mixing it with the culture-medium. 

Intraperitoneal inoculation of mice may also be made with 
J to 1 c.c. of the blood. This may give rise to strepto- 
coccus or pneumococcus septicemia. 

McJunkin's Tube for Collecting' Blood for Cultures. 
*— The blood is drawn from a vein, as for the Wassermann 
reaction, directly into a large test-tube containing an oxalate 
solution, which prevents clotting, but does not inhibit the 
growth of any organisms present. The blood is perfectly 
protected from contamination, so that it can be carried to the 
laboratory, where it can be inoculated into media in tubes 
and flasks, and, in addition, be plated. The method sim- 
plifies the technique of obtaining blood-cultures by eliminating 
the necessity of carrying much apparatus to the bedside, 



426 



PA THOL O GICAL TE CHNIQ UE. 



where inoculations are always made under difficulties, and 
by rendering it easily possible to take blood-cultures from 
several patients on one trip. 

The preparation of the tube is simple. In a test-tube (a) 
which measures 20 by 200 mm. (Bausch and Lomb, No. 



\ 



— 



testtube--- 



V 



Fig. 146. — Mcjunkin's tube for collecting blood for cultures. 

16,920) there are placed 15 c.cm. of a solution which con- 
tains 2 grams of ammonium oxalate and 6 grams of sodium 
chlorid to the liter of distilled water. This is marked (b) in 
the figure. Cotton 4 cm. wide (c) is now wrapped around 



CLINICAL PATHOLOGY. 427 

the rubber tubing (a), which is 150 mm. long, with a i-mm. 
wall and a 3-mm. lumen, and into the upper end of this 
tubing there is inserted a 19-gauge needle 1 inch long (e). 
The needle is capped by inserting into the upper end of the 
large tube a smaller one (/"), which measures 12 by 100 
mm. (Bausch and Lomb, No. 16,920), and which has wrapped 
around the lower end a 3 -cm. plug of cotton (g). Both 
cotton plugs should fit snugly. The tube complete is auto- 
claved for twenty minutes at 1 io° C. The needles should be 
fitted with as large a stylet as possible, so as to insure patency 
of the needle. 

After the needle has been inserted into the vein, a few 
seconds are required for the blood to pass through the 
tubing. The lower cotton plug eliminates all chances of 
contamination, and when it is removed in the laboratory the 
upper end of the tube is flamed and the blood removed with 
a 10-c.cm. sterile pipet (/i). The tubing and needle are 
cleansed by forcing through them a few cubic centimeters 
of water from a syringe, after which they are placed in a 
saturated solution of borax. 

Serum Diagnosis of Syphilis by Means of the 
Wassermann and Noguchi Reactions. 1 — The import- 
ance of the so-called Wassermann reaction in syphilis, aside 
from its scientific interest, lies in the fact that it affords, as 
far as we know at present, a method by means of which ac- 
curacy in diagnosis may be increased, treatment better regu- 
lated, and probably a more definite opinion as to prognosis 
arrived at. The true nature of the reaction is still undeter- 
mined. We know that it is a complicated biological re- 
action, based on the phenomenon of complement fixation ; a 
brief consideration of the theory of this phenomenon will 
therefore be necessary before stating the methods of carrying 
out the test. 

In the first place, a few terms that are constantly being 
used will be defined : 

Complement (Alexine). — This is a substance which is found 

1 Written by Drs. 0. S. Hillman and A. M. Burgess. 



428 PATHOLOGICAL TECHNIQUE. 

in all fresh sera; its activity is destroyed by exposure to 
heat at 5 5 or 56 C. for half an hour. Serum treated in this 
way is said to be inactivated, and can be reactivated by the 
addition of another serum containing active complement. 
The sera of various animals differ in their complementary 
activity and also in their fixability, which is another char- 
acteristic that is possessed by complement. Anti-comple- 
mentary action is a property which develops in a serum on 
standing or which may be present to a certain degree at the 
time the serum is drawn. In selecting a serum for the 
Wassermann reaction it is best to choose one which has the 
greatest degree of activity and fixability. It has been found 
that guinea-pig serum fulfils these demands probably better 
than the serum of any other species. 

Amboceptor. — This is a specific reaction product, which 
may be present in any normal serum, and which can be 
produced in the serum of an animal by repeated injections 
(immunization) of cells or substances (erythrocytes, serum, 
egg-albumin, etc.), for which it has no natural amboceptor. 
Amboceptors that are normally present in serum are called 
natural amboceptors ; those which are produced as the re- 
sult of artificial immunization are called immune ambo- 
ceptors. Amboceptors are classified according to the par- 
ticular substances employed in their production ; for example, 
hemolytic amboceptors (also called hemolysins) are those 
that are produced by the injection of red blood-corpuscles 
into an animal ; bacteriolytic amboceptors (bacteriolysins) 
are produced by the injection of bacterial extracts. An am- 
boceptor is specifically defined by prefixing the term " anti- " 
to the name of the particular species employed in its produc- 
tion ; for instance, when sheep's erythrocytes are the immu- 
nizing agent, the amboceptor is designated as an anti-sheep 
hemolytic amboceptor. 

Complement and amboceptor are the two factors necessary 
in the production of serum hemolysis. This can be demon- 
strated by a simple experiment, as follows : immunize a rab- 
bit to human red blood-corpuscles by means of repeated in- 



CLINICAL PATHOLOGY. 429 

jections, thereby producing in the rabbit serum an anti- 
human hemolytic amboceptor. If serum from such a rabbit 
is brought into contact with a suspension of washed human 
red blood-corpuscles, dissolution of the corpuscles or hemo- 
lysis will take place ; if, however, the rabbit serum be heated 
to 56 C. for one-half hour (inactivated), and then corpuscles 
added, no hemolysis will occur. Finally/if normal human 
serum or normal guinea-pig serum be added to the mixture, 
hemolysis will go on as before. These three factors which 
enter into this reaction, namely, the complement, the hemo- 
lytic amboceptor, and the red blood-corpuscles, constitute 
what is called, for the sake of brevity, the hemolytic system. 

The function of the amboceptor in the above reaction, of 
hemolysis is to sensitize or prepare the erythrocytes for the 
action of the complement ; the latter then has the power of 
causing dissolution of the red cells, resulting in a clear red fluid. 
Neither amboceptor nor complement acting alone can pro- 
duce this result. For complete hemolysis a definite ratio 
must exist between the various factors — amboceptor, com- 
plement, and erythrocytes. The requisite strength and pro- 
portion of these three can readily be estimated by titration, 
which will be taken up under Standardization of Reagents. 

Antigens and Antibodies. — Antigens are substances which, 
when injected into a suitable animal, are capable of producing 
in that animal substances called antibodies, the latter thus 
being specific reaction products. Erythrocytes, bacteria, and 
proteins are examples of antigens. Under antibodies are in- 
cluded hemolytic and bacteriolytic amboceptors, agglutinins, 
and precipitins. Antibodies are also found in the serum of 
patients suffering from infections with micro-organisms. In 
typhoid fever, for instance, an antibody is developed in the 
patient's serum as a result of the action of the typhoid ba- 
cillus upon the immunizing mechanism of the body. 

Generally speaking, it may be stated that antigens and anti- 
bodies bear a specific relationship toward one another ; for 
instance, the hemolytic amboceptor produced by injecting a 
rabbit with sheep's red blood-Corpuscles acts with these cor- 



430 PATHOLOGICAL TECHNIQUE. 

puscles only and with no others. The agglutination of typhoid 
bacilli by the serum of the typhoid patient is also an example 
of this intimate connection between antigen and antibody ; 
this fact is made practical use of in the Widal reaction for the 
determination of the typhoid agglutinin (antibody). The 
phenomenon of precipitation is another instance of the visible 
and direct action between antigen and antibody. Both agglu- 
tination and precipitation are dual mechanisms requiring no 
intermediate agent to complete the reaction. 

In syphilis an antibody is supposed to be developed in the 
patient's serum, probably through the action of the treponema 
pallidum. It seems to be doubtful, so far as we know at 
present, whether the antibody in syphilis is actually specific 
or not. However, from a practical standpoint it may be said 
that it is the presence or absence of this so-called syphilitic 
antibody that we seek to demonstrate in the serum diagnosis 
of the disease. 

Complement Fixation. — As stated above, antigen and anti- 
body unite with one another specifically, and, when united, 
acquire the property of fixing or absorbing complement. 
This fact can be best illustrated by the interaction of two 
sets of antigen-antibody combination. Take, for example, a 
suspension of typhoid bacilli (antigen) and bring it into con- 
tact with typhoid serum (antibody); if complement is now 
added, bacteriolysis will result. That complement has been 
fixed or absorbed by this antigen-antibody combination is 
evidenced by the fact that if red blood-corpuscles and their 
specific amboceptor (another antigen-antibody combination) 
be added later, no hemolysis will occur; complement, in 
other words, is not available for hemolysis on account of 
being fixed by the first antigen-antibody combination. This 
is the well-known phenomenon of complement fixation or 
deviation of Bordet and Gengou, upon which the Wasser- 
mann reaction and its various modifications are based. The 
so-called syphilitic antibody present in a patient's serum 
when brought into contact with an antigen is capable of fix- 
ing complement. This reaction is indicated by absence of 



CLINICAL PATHOLOGY. 43 1 

hemolysis when the other two factors of the hemolytic sys- 
tem are added. 

When the reaction was first introduced, it was thought 
that the antigen used in the diagnosis of syphilis was spe- 
cific, as it was then made from the liver of a syphilitic fetus. 
This was the nearest approach obtainable to actual extracts 
of the causative agent, namely, Treponema pallidum. 
It has been conclusively proved that this original antigen 
is not specific, as it has been found that extracts of normal 
livers, as well as other organs, and also certain lecithin 
preparations will fix complement in contact with not only 
luetic sera but also sera from patients infected with lep- 
rosy, yaws, sleeping-sickness, and malaria. The varia- 
bility in the statistics of different writers is probably due 
to the variety of antigens employed, and at present this 
appears to be the principal limitation to the specificity of 
the reaction. 

The aqueous extract of the liver of a syphilitic fetus, which 
is used as an antigen in the original Wassermann reaction, is 
not employed in the following methods on account of its in- 
stability, and, incidentally, on account of the frequent diffi- 
culty of obtaining syphilitic fetuses. The modifications of 
the original test which have been devised depend, for the 
most part, on variations in the source of the antigen and in 
the employment of a different hemolytic system. 

Probably the most important modification is that of 
Noguchi, in which an antihuman hemolytic system is sub- 
stituted for the antisheep and the acetone-insoluble fraction 
of an alcoholic extract of a normal organ (heart, liver, or 
kidney) is used as antigen. Plain alcoholic extracts of 
normal organs and of livers and spleens of syphilitic fetuses 
have been used by many workers in the Wassermann re- 
action, but at the present time the best antigen seems to 
be an alcoholic extract of human heart muscle saturated 
with cholesterin. On account of its stability for long periods, 
this antigen is particularly valuable, especially when reac- 
tions are done at infrequent intervals. 

For purposes of distinction the two following methods 



432 PATHOLOGICAL TECHNIQUE. 

will be designated as the Wassermann and Noguchi reac- 
tions respectively, although, strictly speaking, the name 
Wassermann should be applied to the original method of 
doing the reaction — that is, with the aqueous extract of a 
liver of a syphilitic fetus. 

In the Wassermann reaction the patient's serum is inacti- 
vated in order to destroy the native complement, which is 
present, as a rule, in an appreciable amount. Complement 
of known strength necessary for the reaction is supplied by 
fresh guinea-pig serum. In the Noguchi reaction an anti- 
human hemolytic system is employed, as Noguchi maintains 
that, owing to the presence in human serum of varying amounts 
of natural amboceptor for sheep's corpuscles, many positive 
reactions are rendered negative in the Wassermann test on 
account of an increase in the total amount of amboceptor 
present, thus disturbing the proper proportion between ambo- 
ceptor and complement necessary for complete fixation. It 
is not necessary to inactivate the patient's serum in the 
Noguchi reaction, as the human complement is only very 
slightly hemolytic for corpuscles of the same species, and 
also because the amount of complement present is practically 
negligible, owing to the small quantity of patient's serum 
used for the test. 

A detailed account of the apparatus needed, preparation 
and standardization of reagents, and technique of the two re- 
actions follow. 

Preparation of Reagents. — Glassware.— For the Noguchi 
reaction, small test-tubes 10 cm. in length and I cm. in diam- 
eter are suitable. For the Wassermann test-tubes about 14 
cm. in length and 1.5 cm. in diameter should be used. Six 
or eight I c.c. pipettes, graduated to 0.0 1 c.c, and two or 
three 10 c.c. pipettes, graduated to 0.1 c.c, are required. 
There are also needed glass tubing for the storage of ambo- 
ceptor serum and for making capillary pipettes, rubber pipette 
bulbs, and several large Petri dishes 15 cm. in diameter. 

In performing the Noguchi and Wassermann reactions, 
absolute bacteriological asepsis is not required, but all tubes 



CLINICAL PATHOLOGY. 433 

and pipettes should be thoroughly washed in cold water, 
without soap or chemicals, and dried for three-quarters of an 
hour in a hot-air sterilizer. Tubes in which serum is to 
be preserved for more than forty-eight hours should be 
thoroughly sterilized. 

Centrifuge. — The centrifuge should be one of high sedi- 
menting power, and should carry at least a four-tube head. 

Water Bath. — A water bath (temperature, 37 C.) may be 
used instead of the ordinary incubator, in which case the 
time needed for all reactions and preliminary titrations is re- 
duced one-half, owing to the more rapid warming of the 
tubes and their contents. 

Saline Solution. — The strength of the saline solution to be 
used in the reaction is 0.875 per cent. — i. e., 8.75 grams of 
sodium chloride c. p. to a liter of distilled water. It should 
be autoclaved before use. 

1. Complement. — It is best obtained by bleeding two or 
more guinea-pigs into a large sterile Petri dish and pipetting 
off the clear serum after several hours' standing at room 
temperature. The clot will yield still more serum on stand- 
ing over night in the ice-chest. Complement deteriorates 
rapidly, and should preferably not be used when over 
twenty-four hours old ; 0.02 c.c. of complement is regarded 
as the unit for use in Noguchi's method. For the Wasser- 
mann test, as here described, O.I c.c. of undiluted comple- 
ment is used as the unit. The variations in the activity and 
fixability of the serum from different guinea-pigs make it ad- 
visable to use a mixture of serum from at least two animals. 

2. Amboceptor. — A. Antihuman. — Both the rabbit and the 
guinea-pig furnish strong antihuman amboceptor, which 
easily activates guinea-pig complement. Of the two, the 
rabbit is preferable on account of its size. Noguchi has 
shown, however, that guinea-pig amboceptor is even stronger 
in activating guinea-pig complement than is that produced in 
the rabbit. Guinea-pigs, therefore, are to be preferred in 
case there is difficulty in obtaining human blood in sufficient 
quantities for the inoculation of rabbits. 

28 



434 PATHOLOGICAL TECHNIQUE. 

Human corpuscles for injection may be obtained by defi- 
brinating the blood when collected, or by adding to it o.i 
per cent, sodium oxalate to prevent clotting. (In using the 
latter method one part of a I per cent, solution of sodium oxa- 
late should be added to nine parts of blood and the mixture 
thoroughly shaken.) The blood should then be strained 
through sterile gauze into sterile centrifuge tubes, the vol- 
ume marked on the outside of each tube, and a large amount 
of saline solution added. After stirring the contents of each 
tube with a sterile glass rod they should be centrifugalized 
until the corpuscles have all sedimented. The supernatant 
fluid is then decanted or siphoned off and the washing re- 
peated at least three times. Sufficient saline is then added 
to each tube to make its contents equal in volume to the 
original blood. The corpuscles are then ready for injection. 

Rabbits should receive five intraperitoneal injections at in- 
tervals of from three to five days (preferably four). The 
following amounts of washed corpuscles should be used: 5, 
8, 12, 15, and 20 c.c. Nine days after the last injection the 
animal should be killed and its serum tested for amboceptor. 
If guinea-pigs are used, four injections of 2, 3, 5, and 7 c.c. 
are given into the peritoneal cavity. The intervals are the 
same as in the case of rabbits. 

The blood of the rabbit, nine days after the last injection, 
may be collected as follows : The animal is etherized, the 
carotid artery dissected out, and a thread passed beneath it ; 
then, with a pair of sharp scissors, the artery is snipped half 
through and the blood allowed to flow into a large, sterile 
test-tube. It is kept at room temperature for several hours 
and then is placed on ice, and the clear serum is pipetted off 
every day for three or four days. 

The different specimens of serum thus obtained are mixed 
and heated to 56 C. for half an hour, to destroy comple- 
ment. After titration (see Standardization of the Ambo- 
ceptor) the serum is hermetically sealed in glass tubes, which 
are to be opened as used. It should be kept in the ice-chest, 
or, better, frozen. 



CLINICAL PATHOLOGY. 435 

B. Antisheep. — Antisheep amboceptor is prepared in the 
same way as is the antihuman. As sheep's blood can ordi- 
narily be obtained in sufficient quantities, rabbits are injected 
instead of guinea-pigs. Blood obtained at an abattoir can 
conveniently be collected and defibrinated in a sterile glass 
jar containing glass beads. The method of washing and the 
schedule for injection are identical with that already described. 

3. Antigen. 1 — For the Wassermann reaction the antigen 
is prepared as follows: 

Human heart muscle freed from excess of fat, is put 
through a meat grinder and mixed with nine times its 
weight of cubic centimeters of 95 per cent, ethyl alcohol. 
The mixture is placed in the incubator for about ten days 
and is shaken from time to time. It is then filtered and 0.4 
per cent, of cholesterin added. It is then again placed in the 
incubator for two or three days and shaken from time to 
time. When the cholesterin is all dissolved the mixture is 
ready for use as antigen and keeps indefinitely. For use 
it is diluted with 4 parts of normal salt solution, which 
should be added slowly and in small quantities at a time, 
with thorough mixing, in order to obtain an emulsion of 
proper character. 

For the Noguchi modification the antigen is prepared as 
follows : 

Bovine heart (which can be particularly recommended) 
or human heart or liver is finely minced, weighed, and 



1 For the original Wassermann an aqueous extract may be prepared as fol- 
lows: Take the liver of a syphilitic fetus and cut it up into fine pieces; mix 
one part with four parts of salt solution (0.85 per cent.), to which 5 per cent, 
carbolic acid is added in the proportion of 0.5 per cent. Shake the mixture 
in a dark bottle for four hours by means of a shaking machine. Centrifugalize 
the mixture and decant off the supernatant fluid. Keep in the refrigerator in 
a dark, rubber-corked bottle. After a few days a precipitate falls to the 
bottom of the bottle. The clear supernatant fluid is used as antigen. 

An alcoholic extract of normal liver or heart, or of a liver of a syphilitic 
fetus, may be made as follows : The tissue selected is taken in the proportion 
of I gram to 5 c.c. of absolute alcohol. Cut the tissue up into small pieces 
and grind it with sand to facilitate extraction. Extract for five days at room 
temperature in an ordinary glass jar, shaking the jar occasionally. Allow it to 



436 PATHOLOGICAL TECHNIQUE. 

covered with ten times its weight of 95 per cent, alcohol. 
The mixture is placed in the incubator at 37 C. for a 
week, and is stirred or shaken vigorously once or twice a 
day during this time. The mixture is then filtered through 
filter-paper and evaporated to dryness at room tempera- 
ture. This may be accomplished by exposing the liquid 
in large evaporating dishes to a current of air from an elec- 
tric fan. The residue is dissolved in a large quantity of 
ether, the solution filtered, and the clear filtrate evaporated 
to dryness. This residue is again taken up with as small a 
quantity of ether as is needed to dissolve it. To the ethereal 
solution five volumes of acetone are added and the whitish 
precipitate is allowed to settle. Most of the supernatant 
acetone is then removed by decantation or siphonage, and 
the remainder is allowed to evaporate. A mass of sticky 
yellow to brown material remains. This mass is weighed 
and dissolved in a sufficient- quantity of methyl alcohol to 
make a 3 per cent, solution. This is the stock antigen 
solution. 

Measured amounts of this alcoholic solution may be kept 
hermetically sealed in test-tubes, which are to be opened as 
used. It is stable at room temperature. For use, one part 
of it should be combined with nine parts of normal saline, 
making an opalescent emulsion. Inasmuch as in this form 
the antigen is often not stable for more than two weeks, it is 
advisable to keep the alcoholic preparation sealed in small 
amounts. For this purpose small test-tubes, such as are 
used in the Noguchi test, are convenient. 

. 4. Patients Scrum. — Blood may easily be obtained in most 
cases from an arm vein. For this purpose ordinary sterile 
hypodermic needles are suitable. After the arm has been 
scrubbed with soap and water and alcohol, a tourniquet is 
applied to the upper arm and the needle is inserted into a 

stand about two weeks. Use the clear, supernatant fluid for antigen, diluting 
it to the extent desired with normal salt solution. In hot weather keep the jar 
in the refrigerator. 

Whichever antigen is used, it must first be titrated to determine its strength 
and suitability. 



CLINICAL PATHOLOGY. 437 

distended vein. The blood is allowed to drop from the 
needle into a sterile test-tube. In this way from 5 to 
15 c.c. can ordinarily be obtained with little difficulty. 
Blood may also be taken from the ear or finger. 

The blood is allowed to stand at room temperature, 
and on separation of the clot the clear serum is pipetted off. 
If the clot adheres to the side of the tube, it should be gently 
separated by a sterile glass rod or platinum needle. Serum 
from a specimen to which sodium oxalate or other chemical 
has been added is unsuitable for the test. 

5. Corpuscle Suspension. — The human corpuscle suspen- 
sion may be prepared by washing human corpuscles, as de- 
scribed under preparation of the amboceptor, and making a 
10 per cent, suspension. A simple method is to fill a grad- 
uated centrifuge tube with 9 c.c. of saline, drop in 1 c.c. of 
blood from one's own finger, wash twice, and make up to 10 
c.c. with saline. 

For the Wassermann test a 5 per cent, suspension of 
washed sheep's corpuscles is used. This is prepared by 
adding nineteen parts of saline to one of washed corpuscles 
(made up in volume to equal the original blood). 

No corpuscle suspension should be used when over seventy- 
six hours old, or when there is any trace of hemolysis. 

Standardization of Reagents. — /. Amboceptor — Noguchi. — 
Into two test-tubes put 0.4 and o. 1 c.c. of immune serum. 
Make the volume in each case up to 10 c.c. with saline. 
Next, to a series of ten small test-tubes add graded amounts 
from these tubes as follows : 

Small Tubes. 

Tube 1. — 4 per cent, amboceptor ( 1 ) .4 c.c. = .016 undil. amboceptor serum- 



Tube 2. — I per cent. 



(2) .3 " 


= .012 


(3) .2 - 


= .008 


(4) -I " 


= .004 


(5) -3 " 


= .003 


(6) .2 « 


= .002 


(7) .1 " 


= .001 


(8) .08 " 


= .0008 


(9) -05 " 


= .0005 


(10) .03 - 


= .0003 



438 PATHOLOGICAL TECHNIQUE. 

To all tubes add o. I c.c. of a 20 per cent, solution of fresh 
guinea-pig complement and 0.1 c.c. of a 10 per cent, suspen- 
sion of washed human corpuscles. Make the contents of 
every tube up to 1 c.c. with saline. Incubate two hours. 
The smallest amount of amboceptor which has caused com- 
plete hemolysis at the end of this time is the amboceptor 
unit. 

Each time the Noguchi test is performed the amboceptor 
should be retitrated, so that the unit may be accurately de- 
termined with regard to the complement and corpuscles 
actually in use. For example, if the titre (standard of 
strength) of the amboceptor was shown in the original titra- 
tion to be 0.00 1 each time the test is performed, a titration 
such as the following should be carried out : 

Amboceptor diluted to 1 per cent. 



1. 0.15 c.c. I per cent, amboceptor. 

2. 0.12 c.c. 1 " " 

3. O.I c.c. l u " \ + * 

4. 0.08 c.c. I " " 

5. 0.05 C.C. I " "J 



1 l unit of complement (o. I c.c 20 
per cent, complement and 0.1 
c.c. 10 per cent, washed human 
corpuscles) to every tube. 



Make the volume of every tube up to 1 c.c. Incubate two 
hours. This can conveniently be carried out so that its in- 
cubation is finished at the same time as the first incubation 
of the test. 

If it is preferred to use the amboceptor dried on paper, 
the following method is recommended by Noguchi : Cut 
filter-paper (Schleich & Schvill, No. 597) into squares 10 x 10 
cm. and place in a large Petri dish. Pour over them the 
amboceptor serum (about 10 c.c. for ten squares), saturating 
as evenly as possible. Absorb any excess with more filter- 
paper. Separate as quickly as possible and dry on clean, 
unbleached muslin for five or six hours. Cut into strips 5 
mm. in width. In this form the amboceptor may be titrated 
by adding various lengths to a series of tubes containing 
complement and corpuscles, as before. Noguchi recom- 



CLINICAL PATHOLOGY. 439 

mends using the amboceptor in this form without a prelim- 
inary titration each time the test is performed. 

Wassermann.— The titration of antisheep serum may be 
carried out as follows : Make 4 per cent, and 1 per cent, 
solutions, as in the case of the antihuman serum. Then, to 
a series of large test-tubes, add the same graded amounts of 
the diluted serum (see Titration of Antihuman Amboceptor). 
To each tube add 0.1 c.c. of undiluted guinea-pig serum and 
1 c.c. of 5 per cent, washed sheep's corpuscles. Make up 
the volume of each tube to 3 c.c. Incubate two hours. 
The unit of amboceptor is contained in the smallest amount 
of serum which has produced complete hemolysis at the 
end of this time. As in the case of the Noguchi reaction, 
a preliminary titration of the amboceptor should always 
be carried out in connection with the performance of the 
test. 

2. Antigen. — The antigen emulsion, to be suitable for use, 
must not possess any appreciable hemolytic or anticomple- 
mentary qualities, and must possess sufficient antigenic 
strength to allow its use in conveniently small quantities. 

I. Noguchi system. 

The following tests are recommended by Noguchi : 

Tube 1. 

0.4 c.c. antigen emulsion. Complete absence of hemolysis at the 

o.l c.c. 10 per cent, human corpuscles. end of this time indicates that the 

Incubate two hours. antigen emulsion does not possess 

any appreciable hemolytic property. 



Tube 2. 

0.4 c.c. antigen emulsion. Complete hemolysis indicates that the 

O.i c.c. 40 per cent, guinea-pig serum. antigen is not inherently anticow 

Make volume I c.c. with saline. plementary. 

Incubate one hour. 

Add — 2 units of amboceptor and o.l 
c.c. 10 per cent, corpuscle suspen- 
sion. 

Incubate two hours. 



44-0 PATHOLOGICAL TECHNIQUE. 

Tube j. 

I unit of syphilitic antibody. 1 Absence of hemolysis shows that 0.02 

O.02 c.c. antigen emulsion. c.c. of the emulsion has sufficient 

O. I c.c. 40 per cent, guinea-pig serum. antigenic strength to fix completely 

Make up to I c.c. two units of complement. 

Incubate one hour. 
Add — 2 units of amboceptor and o.l 
c.c. 10 per cent, corpuscle suspen- 
sion. 
Incubate two hours. 

An emulsion which fulfils the above requirements is suit- 
able for use. The dose to be used in the Noguchi test is 
O.I c.c. (at least 5 units). 

2. Wassermann System. — The titration of the antigen for 
use in the Wassermann may be carried out in exactly the 
same way, using double the amount of the emulsion and the 
amounts of serum, complement, corpuscles, etc., recom- 
mended under " Technique of the Wassermann Test." 

Technique of the Noguchi Reaction. — 1. Set up a pre- 
liminary amboceptor titration (see Standardization of Am- 
boceptor) and place in incubator. 

2. Set up two rows of small test-tubes as follows : 

(a) Two tubes (one in front row and one in back row) 
for every specimen of serum to be tested. 

{b) A similar set for a positive control serum. 
(c) A similar set for a negative control serum. 

3. Add: 

(a) To both tubes in every set 2 capillary drops of the 
serum to be tested in that set. (Of cerebrospinal fluid use 
0.2 c.c.) 

(J?) To all tubes 0.1 c.c. of 40 percent, guinea-pig serum. 

1 The unit of syphilitic antibody, so called, may be determined by ascer- 
taining the highest dilution of a known positive serum, one drop of which will 
completely fix the complement in the ordinary test (see Technique of Test). 
A series of tubes containing various dilutions of the serum are prepared and 
one drop from each is tested. The antigen to be used in this preliminary de- 
termination should be an antigen of proved strength, or, if such is not to be 
had, 0.4 c.c. of the emulsion to be tested, which amount may fairly be esti- 
mated to contain an excess of antigenic property. 



CLINICAL PATHOLOGY. 44 1 

{c) To front row only, 0.1 c.c. antigen emulsion. Make 
the volume of every tube I c.c. 

Incubate one hour at 37 C. 

4. Add to every tube 2 units of amboceptor and 0.1 c.c. 
10 per cent, washed human corpuscles. 

Incubate two hours at 37 C. 

At the end of two hours the results may be read. 

Technique of the Wassermann Reaction. — 1. Set up a 
preliminary titration of the amboceptor and place in incu- 
bator. 

2. Set up two rows of large test-tubes as follows : 

(a) A set of two tubes (one in front row and one in 
back) for each serum to be tested. 

(b) A set for a positive control serum. 

(c) A set for a negative control serum. 

3. Add : 

(a) To both tubes in every set 0.2 c.c. of the serum to be 
tested in that set. (Of cerebrospinal fluid use 1 c.c.) 

(b) To all tubes o. 1 c.c. of undiluted complement. 

(c) To front row only, 0.2 c.c. antigen emulsion. Make 
volume of every tube up to 1.5 c.c. 

Incubate one hour at 37 C. 

4. Add to all tubes 2 units of amboceptor and 1 c.c. of a 
5 per cent, suspension of sheep's corpuscles. 

Incubate two hours at 37° C. 

At the end of two hours the results may be read. 

Interpretation of Results. — The judgment of a positive 
or negative reaction depends upon the absence or presence 
of hemolysis at the end of the incubation period or within a 
few hours (two to five) after the tubes have been standing at 
room temperature. As a rule, the reactions that are going to 
be clean-cut can be read at the end of the incubation period. 

There is no difficulty in deciding frank positive or nega- 
tive reactions, but difficulty is sometimes encountered in in- 
terpreting the gradations that are not infrequently seen where 
varying degrees in the intensity of hemolysis are present. 
Certain points are to be observed w r ith both methods before 
the reactions are finally read. These are as follows : 



442 PATHOLOGICAL TECHNIQUE. 

1. The back row of tubes should be completely hemolyzed 
at the end of the incubation period in order to make sure that 
the hemolytic system is working satisfactorily, and, also, that 
the sera which are being examined are not anticomplementary. 

2. The negative control should show complete hemolysis 
in both front and back tubes for the same reasons. 

3. The positive control should show no hemolysis in the 
front tube, where the antigen-antibody combination has oc- 
curred, but in the back tube hemolysis should be complete for 
the same reasons noted under " 1." 

These three conditions being fulfilled, the test reactions 
may now be read. 

In the Wassermann test, positive reactions are indicated at 
the end of the incubation period by absence of hemolysis in 
the front tubes. When the tests are first taken out of the 
incubator (or water-bath), a pink to pinkish-gray cloud ex- 
tends up from the bottom of the tubes and may take in almost 
the entire bulk of fluid present. This is due to suspension 
of a certain number of red blood-corpuscles in the fluid. 
After standing a couple of hours, however, the corpuscles 
settle out in a sharp layer at the bottom of the tubes which 
are strongly positive, leaving a clear, colorless, supernatant 
fluid. In the Noguchi modification a similar condition is ob- 
served in positive reactions, but owing to the smaller amount 
of corpuscles and fluid, settling takes place more rapidly than 
in the Wassermann tubes. Reactions are sometimes en- 
countered with the Noguchi test, in which the corpuscles 
settle out in a sharp layer, but the supernatant fluid, instead 
of being perfectly colorless, is slightly red colored. These 
reactions, however, are to be regarded as positive, although 
not strongly so. Negative reactions by both methods result 
in a perfectly clear red fluid, with complete hemolysis and 
disappearance of all the red blood-corpuscles. 

Weakly positive reactions, are indicated in both tests by 
various degrees of coloring of the supernatant fluid, and by 
variations in the amount of red blood-corpuscles in the bot- 
toms of the tubes. 

In the Noguchi test, weak reactions are of special signif- 



CLINICAL PATHOLOGY. 443 

icance, as they give distinct information regarding the 
strength of the so-called syphilitic antibody; therefore, in 
reading these weak reactions, it is important to note the ex- 
tent to which hemolysis has progressed. The Noguchi test 
is of particular value in cases that are undergoing, or have 
previously received, treatment. 

The presence in human serum of variable amounts of nat- 
ural amboceptor for sheep's corpuscles renders the Wasser- 
mann test less valuable as an indicator of the strength of the 
syphilitic serum. 

In the Wassermann test, reactions which do not show com- 
plete absence of hemolysis are regarded as weakly positive ; 
those which show well advanced hemolysis are called very 
weak or doubtful, and, if possible, these reactions should be 
done over again, using fresh patient's serum and carefully 
titrating the reagents. 

From the foregoing statements it can be seen that in ex- 
amining sera, the serologist should always be informed 
whether or not antisyphilitic treatment has been administered, 
in order that he may be able to interpret the results of the 
reactions with more value to the clinician. 

It has been found helpful to send out with reports of 
Wassermann reactions a printed slip containing the following 
statements : 

It has become customary to recognize three degrees of the 
positive reaction and to indicate them with one, two, or three 
plus signs. The interpretation to be placed on these different 
degrees of the positive reaction is as follows : 

Positive -f- is less than a 10 per cent, inhibition of 

hemolysis and should not be considered 
of any diagnostic value except in a case of 
syphilis under treatment ; then it calls for 
further active treatment. (Some workers 
with the Wassermann test prefer to have 
the lowest degree of positive reaction in- 
clude as high as 50 per cent, inhibition of 
hemolysis.) 






444 PATHOLOGICAL TECHNIQUE. 

Positive -| — |- is an inhibition of hemolysis, varying be- 
tween 10 and 90 per cent. It should be 
considered as a positive diagnosis of syphilis 
only in connection with strong clinical evi- 
dence. In a case of syphilis which has 
been treated it calls for further active 
treatment. 

Positive -\ — | — f- is complete inhibition of hemolysis, and is 
to be considered as a positive diagnosis of 
syphilis. 

The Wassermann reaction is not absolutely specific for 
syphilis. 

A positive reaction, not due to syphilis, is frequently ob- 
tained in yaws, trypanosomiasis, and leprosy, and according 
to some authorities may also be obtained in rare instances in 
other conditions, such as scarlet fever, malaria, lobar pneu- 
monia, diabetes mellitus, and immediately following ether 
anesthesia. 

A negative reaction is obtained in many cases of syphilis' 
during treatment with salvarsan or mercury, and may occur 
in alcoholics. 

Antisyphilitic treatment must be interrupted for two months 
before a negative Wassermann reaction can be considered to 
have any diagnostic value. 

The blood to be tested by the Wassermann reaction must 
be used as fresh as possible, before hemolysis due to warmth 
or bacterial contamination has taken place. The blood can 
be kept in good condition for the test for twenty-four to 
forty-eight hours if placed in a refrigerator. 

A Modification of the Wassermann Reaction (J. H. Wright). 
— This modification employs as complement and hemolytic 
amboceptor unheated human serum or a mixture of sera 
which, on the day of use, have been found capable of hemo- 
lyzing a certain amount of sheep corpuscles in the presence 
of a certain amount of antigen. 

This preliminary procedure of the method may be de- 
scribed as follows: Select several unheated sera which are 



CLINICAL PATHOLOGY. 445 

probably not syphilitic. Place 0.1 c.c. of each serum in a 
test-tube, then add to each tube 0.1 c.c. of antigen freshly 
diluted for use, as in the Wassermann test, and, after five 
minutes, 0.5 c.c. of the sheep corpuscle suspension diluted 
with 4 parts of normal salt solution. Put all the tubes in 
the water-bath and after fifteen minutes note the tubes in 
which complete hemolysis has occurred. Mix the corre- 
sponding sera for use as complement and hemolyzing am- 
boceptor in the test to be set up as described below. Among 
every ten sera several will generally be found suitable for 
use. They may retain their hemolyzing power for some 
days. 

To make the test, place in a test-tube, in the order named, 
0.1 c.c. of the inactivated serum to be tested, 0.1 c.c. of 
the mixed active hemolyzing sera, 0.1 c.c. of the diluted 
antigen, and, after five minutes' intubation, 0.5 c.c. of the 
diluted suspension of sheep corpuscles. Incubate the mix- 
ture and read as in the Wassermann test described above. 
If hemolysis be inhibited, a control test without antigen 
should be made. 

It is important that the sheep corpuscle suspension be 
added within a very few minutes after the serum and the 
antigen have been combined in the tube. 

Comparative tests have shown a very close agreement in 
results with the regular Wassermann reaction. 

The principles upon which this modification is based were 
first called to my attention by the observations of C. J. 
Bartlett and A. L. O'Shansky. 1 

The Complement-fixation Test in Gonorrheal In- 
fections. — The method and technique are essentially the 
same as that of the Wassermann test, except that the antigen 
is different. 

Preparation of the Antigen. — The growth from twenty- 
four-hour cultures of the gonococcus on hydrocele agar slants 
is scraped off with a platinum loop and suspended in a 0.5 
per cent, solution of carbolic acid in distilled water. The 

1 "A Modified Wassermann Technique Based Upon the Rapid Fixation 
of Complement Present in Human Serum," by C. J. Bartlett and A. L. 
O'Shansky, Jour. Lab. and Clin. Med., 191 7, iii, 118. 



446 PATHOLOGICAL TECHNIQUE. 

proportion of cocci to fluid should be the entire surface 
growth on the ordinary slant culture in a three-quarter-inch 
test-tube to about 5 c.c. of the fluid. The suspension is to 
be kept at room temperature and to be shaken from time to 
time during a week, after which it should be heated to 56 
C. for an hour and centrifugalized thoroughly. To the su- 
pernatant fluid is then added sufficient 9 per cent, sodium 
chlorid solution to make its content in that salt 0.9 per 
cent. This is the antigen. Every time it is used it must be 
titrated to determine its anticomplementary dose, and one- 
half of the largest amount which does not inhibit hemolysis 
is to be used in the test. The addition to the antigen of 
one drop of a saturated alcoholic solution of cholesterin for 
• each cubic centimeter of antigen just before use may be. 
found to increase its fixing power. 

The suspension of the cocci in the carbolic acid water re- 
tains its fixing power for months, and it may be kept on 
hand in quantities as a. stock from which, as needed, small 
quantities may be taken, centrifugalized, and the proper 
amount of sodium chlorid added. 

Considerable -variation in the fixing power of different anti- 
gens is observed, and it is advisable to use two or three anti- 
gens from different strains of gonococci in each test or a 
mixture of antigens from several strains. 

The Complement-fixation Test in Echinococeus 
Infection. — The method and technique are the same as 
those for the Wassermann test, except that the cyst fluid, or 
an alcoholic extract of the cyst walls, is used as the antigen. 
The cyst fluid should be clear, and to it should be added 
carbolic acid in the proportion of 0.5 per cent, to preserve it. 

The extract of the cyst walls is prepared by grinding them 
up with sand and extracting the mass with absolute alcohol, 
in the proportion of about one part cyst substance to five of 
alcohol, for several days. 

The anticomplementary dose of the fluid or of the extract, 
the latter diluted 1 to 4 with normal salt solution, is to be 
determined by titration, and one-fourth of this quantity is to 
be used in the test. 

These antigens are said to keep for months. 



CLINICAL PATHOLOGY. 44/ 

EXAMINATION OF THE CEREBROSPINAL FLUID. 

I/Umbar Puncture. — The diagnostic value of lumbar 
puncture has been sufficiently demonstrated. Not only is 
it possible to diagnosticate inflammation of the meninges, 
but the character and cause of the inflammation may usually 
be demonstrated if the examination of the fluid is properly 
performed. In a number of cases of general infection in 
which there was no inflammation of the meninges a diag- 
nosis has been made by means of cultures taken from the 
cerebro-spinal fluid. Finally, a number of cases of hemor- 
rhage into the brain and spinal canal have been diagnosti- 
cated by lumbar puncture. 

The operation and the subsequent examination of the fluid 
should be as carefully performed as any other bacteriological 
investigation in order to obtain accurate results. The back 
of the patient and the operator's hands should be made 
sterile. The needle should be boiled for ten minutes. The 
patient should lie on the right side, with the knees drawn up, 
and with the uppermost shoulder so depressed as to present 
the spinal column to the operator. This position permits the 
operator to thrust the needle directly forward rather than 
from the side. An antitoxin needle 4 cm. in length, with a 
diameter of 1 mm., is well adapted for infants and young 
children. A longer needle is necessary for adults and chil- 
dren over ten years of age. 

Aspiration of the fluid is not necessary, but some opera- 
tors prefer to attach a hypodermic syringe to the needle to 
afford a better grasp for the hand. In this case the syringe 
would have to be detached to allow the fluid to flow. The 
additional manipulation, and possibly the defective steriliza- 
tion of the syringe, might impair the subsequent bacterio- 
logical examination. 

The puncture is generally made between the third and 
the fourth lumbar vertebrae ; sometimes between the second 
and third. The thumb of the left hand is pressed between 
the spinous processes, and the point of the needle is entered 
about 1 cm. to the right of the median line and on a level 



448 PATHOLOGICAL TECHNIQUE. 

with the thumb-nail, and directed slightly upward and in- 
ward toward the median line. Care must be exercised to 
prevent the point of the needle from passing to the left of 
the median line and striking on the bone. At a depth of 3 
or 4 cm. in children and 7 or 8 cm. in adults the needle en- 
ters the subarachnoid space, and the fluid flows usually by 
drops. If the point of the needle meets with a bony obstruc- 
tion, it is advisable to withdraw the needle somewhat, and to 
thrust again, directing the point of the needle toward the 
median line, rather than to make lateral movements, with the 
danger of breaking the needle or causing a hemorrhage. 
The smallest quantity of blood obscures the macroscopic 
appearance of the fluid by rendering it cloudy. The fluid is 
allowed to drop into an absolutely clean test-tube which pre- 
viously has been sterilized by dry heat to 150 C. and stop- 
pered with cotton. The fluid should be allowed to drop into 
the tube without running down the sides. From 5 to 15 c.c. 
of fluid is a sufficient quantity for examination. 

In meningitis there is always an exudation of cells which 
makes the fluid more or less cloudy. The degree of cloudi- 
ness is to some extent proportionate to the amount and char- 
acter of the exudation. In tubercular meningitis the amount 
of cellular exudation is sometimes so slight that the fluid 
appears clear unless examined carefully. 

Cultures on blood-serum and cover-glass preparations 
should be made from the fluid. In most cases this is best 
done from the sediment thrown down by the centrifuge. It 
is of great importance that the tube of the centrifuge should 
be clean and sterile. If tubercular meningitis is suspected, 
a guinea-pig may be inoculated with the sediment. 

The cover-glass preparations, after drying in the air, are 
best stained with Wright's blood-stain (see page 418). This 
reveals the characters of the cells very clearly and stains 
any bacteria that may be present, with the probable excep- 
tion of the tubercle bacillus. 

A predominance of polynuclear leucocytes in the sedi- 
ment means non-tubercular meningitis. The infecting bac- 
terium should be sought for and its identity determined. It 



CLINICAL PATHOLOGY. 449 

will generally be the diplococcus intracellularis or the pneu- 
mococcus. A predominance of large and small lymphocytes 
in the sediment indicates the existence of tubercular menin- 
gitis, and cover-glass preparations should be stained for 
tubercle bacilli. It may be necessary to examine twenty or 
more preparations before finding the bacilli, or to inoculate 
a guinea-pig. 

The number of cells in the spinal fluid may be estimated 
by the same method as for white blood-corpuscles. A 
staining fluid which facilitates the enumeration and deter- 
mination of the nature of the cells consists of: 

Acetic acid, 0.2 c.c; 

Methyl- violet, 0.2 gm. ; 

Distilled water, 50.0 c.c. 

Normally there are only one or two cells per centimeter. 
In syphilis there may be fifty to one hundred. 

Protein Increase Tests. — The presence of proteins in 
the fluid may be demonstrated by the production of tur- 
bidity after shaking 1 part of the fluid with 95 per cent, 
alcohol. 

The Ross-Jones method for globulin is to place in a small 
tube 1 c.c. of a saturated aqueous solution of ammonium 
sulphate and overlay this with 1 c.c. of the spinal fluid. 

A globulin increase is shown by the appearance of a 
turbid ring at the junction of the two fluids. 

The Noguchi method for showing increase of globulin is 
as follows: 

To 0.2 c.c. of spinal fluid add 0.5 c.c. of 10 per cent, 
solution of butyric acid in normal salt solution and boil. 
Then add 0.1 c.c. of a 4 per cent, aqueous solution of 
sodium hydrate and boil again. A flocculent precipitate 
appears in from twenty minutes to two hours, when the 
reaction is positive. 

Alzheimer's Method for the Cytological Exami- 
nation of the Cerebrospinal Fluid. — This method is 
especially useful in the diagnosis of general paralysis. The 

29 



450 PATHOLOGICAL TECHNIQUE. 

description of it is taken from a paper by H. A. Cotton and 
J. B. Ayer. The method is as follows : 

1. Lumbar puncture in the usual manner. 

2. 96 per cent, alcohol, in proportion to twice the amount 
of cerebro-spinal fluid, is added drop by drop and well mixed. 

3. Centrifuge the mixture for one hour at high speed in a 
glass tube with conical end. (An ordinary electric urinary 
centrifuge apparatus can be employed, the tube to be well 
stoppered to prevent evaporation.) 

4. The supernatant fluid is poured off, leaving a small co- 
agulum in the bottom of the tube. 

5. Add absolute alcohol — alcohol and ether — ether, each 
separately for one hour, to dehydrate and harden coagulum. 

6. The coagulum can now be gently loosened from the bot- 
tom of the tube by a long needle. The tube is then inverted, 
and the coagulum allowed to fall into the hand by a quick 
tap on the end of the tube. Care must be taken not to 
squeeze or handle the coagulum. The hand is placed over 
a small homeopathic vial, containing thin celloidin, and the 
coagulum allowed to drop into the celloidin, where it remains 
over night (twelve hours usually). 

7. Coagulum placed in thick celloidin which is allowed to 
evaporate slowly. It is then mounted on blocks and sections 
cut 14^ in thickness. 

8. The sections are stained and mounted according to the 
following procedure : 

(a) Remove celloidin by absolute alcohol and ether. 

(b) 80 per cent, alcohol. 
(J) Water. 

(d) Sections are carried on glass or platinum needle into 
a dish of Pappenheim's pyronin-methyl green stain (see p. 
236) and kept in a water-bath at 40 C. five to seven minutes. 

(e) Quickly cool dish in running water. 

(/) Wash off superfluous stain in plain water. 
(g) Absolute alcohol to differentiate — until no more stain 
comes away from section. 

(k) Clear in Bergamot oil. 
(/") Mount in balsam. 



CLINICAL PATHOLOGY. 45 I 

The cells are caught in the coagulum and are nearly evenly 
distributed throughout it. Cross-sections are prepared and 
examined from at least 6 levels in the coagulum. The num- 
ber of cells in ioo fields of a half-inch or similar objective is 
taken as the unit for comparison. A high cell count — that 
is, over ioo cells to ioo fields, the presence of plasma-cells 
and perhaps phagocytes, in a case of suspected general par- 
alysis—is the strongest evidence in favor of this diagnosis. 

lunge's Colloidal Gold Test of the Cerebrospinal 
Fluid for Syphilis of the Central Nervous System. — 
This account of the test is based upon the work of Dr. R. I. 
Lee and Dr. W. A. Hinton with it. 

The test depends upon a change in the color of a solution 
of colloidal gold when it is mixed in certain proportions with 
the cerebrospinal fluid from cases of syphilis and certain 
other pathological conditions of the central nervous system. 

For each test a row of 10 test-tubes, J inch in diameter, is 
set up in a rack. Into the first tube on the left is measured 
with a small pipette 1.8 c.c. of a 0.4 per cent, solution of 
sodium chlorid, and into each of the other tubes 1 c.c. of 
the same solution. Into the first tube on the left is now 
measured 0.2 c.c. of the cerebrospinal fluid, and the contents 
thoroughly mixed by thrice drawing some of it high up into 
the pipette and expelling it. From this tube 1 c.c. of the 
fluid is withdrawn and transferred to the adjoining tube, the 
contents thoroughly mixed as before by means of the pipette, 
and 1 c.c. of this mixture then withdrawn and transferred to 
the next tube in the row, and so on. In this way half the 
contents of each tube is transferred to the next tube and 
mixed until the tenth tube is reached, when 1 c.c. of the con- 
tents of this tube, after thorough mixing, is withdrawn and 
rejected. In the row of 10 tubes there are thus obtained 
the following dilutions of the cerebrospinal fluid, starting 
from the left to right, namely, 1-10, 1-20, 1-40, 1-80, I- 160, 
1-320, 1-640, 1-1280, 1-2560, 1-5 1 20. 

Into each tube is next measured from a 25 c.c. graduated 
pipette 5 c.c. of the colloidal gold solution or " reagent," 
which is described below, and the tubes are shaken. 



452 PATHOLOGICAL TECHNIQUE. 

The "reagent" should be of a color closely approximating 
"old rose" red, with the slightest blue or purplish nuance, 
and have a very slightly yellowish fluorescence. It should 
be clear and transparent. 

With normal cerebrospinal fluid, free from serum or blood, 
no change of color should be apparent in the mixtures in 
any of the tubes. A single tube showing color change indi- 
cates faulty technique. 

With pathological fluids, there is a change of the red color 
in two or more tubes to a more or less marked blue color 
almost immediately. After twelve or twenty-four hours the 
color changes are more marked, and there is more or less 
deposit of a black sediment. After this period, in the tubes 
showing the more intense reaction, the blue color has become 
paler or the fluid may be colorless. 

For recording the appearances in the various tubes six 
degrees of color change are to be recognized, and are ex- 
pressed by numbers, namely : " ±" representing the slightest 
increase in the bluish tint of the reagent, " I " a slightly 
greater change, and so on, " 5 " representing a colorless fluid. 
These color changes, if the technique is right, are gradual 
in the series, i. e. y there should be no marked difference 
between any two adjoining tubes. 

Any form of syphilis of the central nervous system is indi- 
cated by varying degrees of color change ; these changes 
are most marked in the third, fourth, and fifth tubes, counting 
from the left of the row, and corresponding to the dilutions 
of 1-40, 1-80, and 1-160 of the cerebrospinal fluid. Fluids 
from cases which have received intradural injections of sal- 
varsanized serum may give reactions which are different from 
these and may be atypical. As a rule, the fluid of general 
paresis and cerebrospinal syphilis gives stronger reactions 
than that of tabes. 

The fluid from cases of non-specific meningitis and brain 
tumor shows most marked color changes in the seventh and 
1 eighth tubes, corresponding to the dilutions of 1-640 and 
; 1-1280 ; fluids contaminated by blood or serum may also 
produce color changes, most marked in these tubes. Bac- 



CLINICAL PATHOLOGY. 453 

terial contamination of the fluid weakens the reaction and 
may change a positively reacting fluid into a negatively re- 
acting one. 

The preparation of the colloidal gold " reagent " requires 
great care, and the obtaining of a solution which answers the 
requirements as to color and clearness appears to be depend- 
ent to some extent upon conditions beyond the control of 
the operator, so that several lots may have to be made up 
before one suitable for use is obtained. The directions for 
the preparation of the reagent are as follows : 

Heat in a tall Jena glass beaker, of about 2 liters capacity, 
500 c.c. of double distilled water to 6o° C. The beaker is 
to be supported on wire gauze over a large Bunsen flame. 
When this temperature is reached, as shown by the ther- 
mometer, without removing the flame, run in 5 c.c of a 1 
per cent, aqueous solution of yellow crystalline chlorid of 
gold and immediately afterward 5 c.c. of a 2 per cent. aque T 
ous solution of potassium carbonate. Continue the heating 
until the temperature reaches ioo° C, and until the smaller 
bubbles cease to be given off by the boiling fluid. Then run 
in 5 c.c. of a 1 per cent, aqueous solution of formalin, remove 
the beaker from the flame, and stir the fluid. The fluid 
should immediately, or within a minute, assume the " old 
rose " red color, and if suitable for use should have the char- 
acters above described. When cool it is ready to be used as 
the " reagent." If it is not perfectly clear when viewed in a 
test-tube of f inch diameter, or if it is of a distinctly bluish 
tint, it should be rejected. It keeps for weeks. 

Special Precautions to be Observed. — All glassware used 
must be chemically clean. 

The water used for the " reagent," for making up the solu- 
tions and for the final washing of pipettes, flasks, beakers, etc.,_ 
must be double distilled from Jena glass in a still free from 
rubber connections, and it must be kept in Jena glass 
flasks. 

The cerebrospinal fluid must be free from blood and from 
bacterial contamination . Fluids kept bacteria-free have given 
the same reactions for weeks. 



454 PATHOLOGICAL TECHNIQUE. 

The small pipette used for measuring the cerebrospinal 
fluid and its dilutions must be cleansed with distilled water, 
absolute alcohol, and ether before use with each specimen 
of cerebrospinal fluid. 

EXAMINATION OF TISSUES AND FLUIDS. 
Examination of Tissues from Clinical Cases for 

Diagnosis. — Tumors of any size or large pieces of tissue 
present no difficulties. There is plenty of material to ex- 
amine fresh or after fixing in a variety of ways. Frozen sec- 
tions of the fresh tissue are often sufficient. Sometimes it is 
better to harden for an hour or more in formaldehyde and 
then to make frozen sections. Often it is wisest to harden in 
strong alcohol and then to make razor sections or to embed in 
celloidin. The whole process of hardening, embedding, and 
sectioning can easily be carried through in twenty-four hours 
with small pieces of tissue. Embedding in paraffin is sometimes 
preferred. In cutting sections of small pieces it is important 
to mount them if possible, so that the cut sections will show 
proper relations — i. e., vertical sections through the skin, 
uterine mucous membrane, etc. — otherwise confusing pict- 
ures will often be presented. It is important to know, in 
regard to pieces of tissue sent for diagnosis, from what part 
of the body they come. 

A hematoxylin-and-eosin stain will be found the most 
generally useful for hardened sections. 

Uterine Scrapings. — Small pieces may be examined 
fresh in frozen sections or after hardening for one or more 
hours in formaldehyde. Better results are obtained by hard- 
ening in alcohol and imbedding in celloidin or paraffin. 
Where the fragments are small, it is advisable to mass them 
together on a small piece of filter-paper and to harden in 
formaldehyde or in Zenker's solution. The mass can then be 
embedded in celloidin and cut as one piece of tissue, or they 
can be carried through by the gelatin-formaldehyde embed- 
ding method (see p. 54), and cut on the freezing microtome. 
A hematoxylin-and-eosin stain is the best, because the eosin 



CLINICAL PATHOLOGY. 455 

brings out the smooth muscle-fibers prominently, so that any 
invasion of the muscular coat by a malignant growth is more 
readily made out — a valuable help in the diagnosis of malig- 
nant adenoma. 

Examination of Fluids obtained by Puncture. — 
The transudations obtained largely from the serous cavities 
are non-inflammatory in origin. They are usually of a trans- 
parent, pale-yellow color with slightly greenish tint, alka- 
line in reaction, and deposit on standing a slight flocculent 
coagulum. 

The specific gravity, to be taken at room-temperature, 
varies according to the origin of the fluid. According to 
Reuss, it is below 1015 in hydrothorax; below 1012 in 
ascites; below 10 10 in anasarca. 

The amount of albumin in hydrothorax is always under 
2.5 per cent, and in ascites between 1.5 and 2 per cent. 
Microscopically, a few leucocytes, usually fatty degenerated 
and rarely desquamated endothelial cells, are found. 

The exudations are of inflammatory origin, and are also 
generally obtained from the serous cavities. From their 
various microscopic appearances they are divided into serous 
(fibrino-serous), hemorrhagic, purulent, and gangrenous. 
The specific gravity of all is over 1018 ; the reaction is always 
alkaline. On standing they deposit a varying amount of 
sediment. Examinations for organisms should always be 
made. Occasionally a peculiar opalescent layer, due to 
cholesterin crystals, forms on the surface of fluids which 
come from old cases of pleurisy. 

Serous Exudations. — The fluid, which immediately after 
removal is slightly cloudy and yellowish in color, deposits 
more or less quickly a flocculent or dense coagulum. Micro- 
scopically, the coagulum shows a dense meshwork of fibrin 
and numerous polynuclear leucocytes. 

Hemorrhagic Exudations. — The sero-fibrinous exudation 
is colored a lighter or darker red according to the amount 
of blood present. Microscopically, the same elements are 
found as in the serous exudations, plus a marked increase 



456 PATHOLOGICAL TECHNIQUE. 

of red blood-globules, which are usually well preserved, but 
in old exudations may be more or less decolorized. 

Aside from injuries, hemorrhagic exudations are most 
common in connection with tuberculosis and new growths, 
so that their microscopic examination is of much diagnostic 
and prognostic value. 

For the examination for tubercle bacilli see page 348. In 
this form of exudation it is rarely possible to demonstrate 
them. On the other hand, it is not infrequently possible to 
make the diagnosis of a malignant growth, especially of 
cancer, from the examination of the sediment. No cell is 
significant of cancer or other neoplasm, but the occurrence 
of numerous cells which vary greatly in form is suspicious. 
The cells from new growths are often unusually large, up to 
120/i, frequently contain one or more vacuoles, and usually 
lie in clumps. Large drops of fat are also considered sus- 
picious. 

A positive diagnosis can only be made by obtaining bits 
of tissue which show the structure of the new growth, such 
as the atypical alveolar arrangement of the cells in cancer. 

Purulent exudations appear more or less thick and yel- 
low, and deposit a corresponding layer of pus. Microscopic- 
ally, they present no peculiarities other than the organisms 
to which they are due. Among the etiological factors acti- 
nomyces must always be thought of in puzzling cases. 

Putrid exudations occur in the pleural and peritoneal 
cavities in consequence of gangrenous masses breaking into 
them and from stomach or intestinal ulcerations, from new 
growths, occasionally from no clear cause. The fluid result- 
ing from the perforation of a gastric ulcer may show yeast- 
cells and sarcinae, and give an acid reaction. 

Examination of Serous Fluids. 1 — In 1900 Widal and 
Ravaut published a method for the examination of serous 
fluids and gave it the name of cy to diagnosis. Recently 
Jousset has described a method for the detection of the 

1 This section has been written by Dr. Percy Musgrave, who has thoroughly 
tested these methods in the Clinico- Pathological Laboratory of the Massachu- 
setts General Hospital. 



CLINICAL PATHOLOGY. 457 

bacillus tuberculosis, under the name of inoscopy. These 
two methods have been found of much importance in the 
examination of serous effusions as means of determining 
their etiology. 

Cytodiagnosis consists in the examination of the cellular 
elements with reference to the variety of cell which predomi- 




Fig. 147. — Cytodiagnosis. Polynuclear leucocytes and swollen endothelial 
cells in a smear preparation from the centrifugalized sediment of the fluid from 
an acute infectious non-tubercular pleuritis (Percy Musgrave ; photo by L. S. 
Brown). 

nates in the sediment. The originators of this method have 
given us the following formulae : 

1. Predominance of polynuclear leucocytes means an acute 
infectious process. 

2. Predominance of lymphocytes means tuberculosis. 

3. Few cellular elements with a large proportion of endo- 
thelial cells, occurring especially in sheets or plaques, means 
a transudate or mechanical effusion. 

These writers have given us no special formula for cancer, 
but there is reason to believe that cancerous fluids show a 
relatively large number of endothelial cells mixed with a 
larger percentage of lymphocytes than is found in the me- 
chanical effusions, and also that cancerous fluids have a large 
amount of albumin and a high specific gravity. Further 
research, however, on this point is necessary. 



458 



PATHOLOGICAL TECHNIQUE. 



The age of the effusion in the acute infectious variety has 
some modifying effects, for which the reader is referred to the 
original articles. 

In the tubercular variety, although there is usually a high 
percentage of lymphocytes in the first ten days, the poly- 
nuclear leucocytes may predominate, but after the second 
week the formula remains fairly constant. 




Fig. 148. — Cytodiagnosis. Lymphoid cells in a smear preparation from the 
centrifugalized sediment of pleural fluid ; case of tubercular pleuritis (Percy 
Musgrave ; photo by L. S. Brown). 

The so-called secondary tuberculous pleurisy (caused by 
direct extension from a tuberculous focus in the lung) often 
shows a large number or even a predominance of poly- 
nuclear leucocytes, owing to a secondary infection with 
pyogenic bacteria. 

Old mechanical effusions are occasionally encountered 
where little endothelium is seen, and the cells found are 
almost exclusively lymphocytes. 

Method. — The fluid should be drawn with the usual 
aseptic precautions into sterilized flasks or tubes. If it is 
clotted, it should be shaken until the clot is thoroughly con- 
tracted, and the clot, or all clots of large size, should be 
removed. 



CLINICAL PATHOLOGY. 



459 



Place the fluid in centrifuge tubes and centrifugalize for 
five minutes at least. 

Decant the supernatant fluid gently 'at first, and when a 
small amount only remains, invert the tube for about two 
seconds. A few drops only will be left. 

With a small platinum loop stir the sediment thoroughly, 
rubbing the sides of the glass to remove adherent portions. 
When the sediment is thoroughly mixed with the few drops 




Fig. 149. — Cytodiagnosis. Endothelial cells in a smear preparation from 
the centrifugalized sediment of a transudate or mechanical effusion (Percy Mus- 
grave ; photo by L. S. Brown). 

of fluid remaining after decantation, remove a drop of the 
mixture with the platinum loop and make a cover-slip smear. 
Allow this to dry spontaneously or by very gently heating. 
Heating at the boiling-point will spoil the preparation. 

Cover the preparation with a staining fluid made as fol- 
lows : 

Wright's blood-stain, 3 parts ; 

Pure methyl-alcohol, I part. 

Allow to remain on the preparation twenty to forty-five 
seconds, then dilute it with 8 to 10 drops of water and allow 
this mixture to stand one to two minutes. 

Wash very gently, preferably by flooding the slide with a 
dropper. Do this four or five times, allowing the water to 
remain on the slide a few seconds each time. Vigorous or 



460 PATHOLOGICAL TECHNIQUE. 

forcible washing will destroy the film and spoil the prepara- 
tion. 

Dry the preparation by holding it between the thumb and 
forefinger and waving it through the Bunsen or alcohol 
flame. Do not attempt to blot the preparation or heat it 
above the temperature which the fingers will bear. 

Mount in xylol-balsam and examine with an oil-immersion 
lens. 

Inoscopy is practised as follows : 

1. The fluid should be drawn with asceptic precautions 
into sterilized flasks (Erlenmeyer flasks preferably), and at 
least 100 c.c. should be taken, although results may some- 
times be obtained with much smaller amounts. Allow the 
fluid thus taken to clot. 

2. Shake the fluid gently to contract the clot as much as 
possible, and then wash it, on a piece of sterile linen or fine 
gauze wrapped over the end of a funnel, until all the serum 
is washed away. 

3. Remove the clot or clots with a sterile spatula and 
place in a small flask with sufficient of the following fluid to 
digest it : 

Pepsin, 2 gm. ; 

Pure glycerin 1 1 T ^ „ „ . 

Strong HCl T^ X °**" 

Sodium fluorid, 3 gm. ; 

Distilled water, 1 000 c.c. 

The amount of this fluid necessary will vary, of course, 
with the size of the clot to be digested, but 20 or 30 c.c is 
sufficient in most cases. A freshly prepared pepsin — HCl 
solution — apparently 'serves as well as the above fluid. 

4. Place the above preparation in the incubator or oven 
until the clot is digested. A temperature of 37 C. for two 
or three hours will suffice, but the time is shortened if kept 
at a temperature of 50 C. 

5. When the clot has disappeared, pour the mixture into 
centrifuge tubes and centrifugalize for five to ten minutes. 
Decant the supernatant fluid as described under cytodiag- 
nosis (see p. 45$). 



CLINICAL PATHOLOGY. 46 1 

6. Make a cover-slip preparation and stain it for tubercle 
bacilli. Care, however, should be taken not to decolorize 
too long — one-half to three-quarters of a minute with Gab- 
bet's solution is sufficient. Dry and mount. 

The majority of the bacilli found by this method are 
shorter and broader, as a rule, than the tubercle bacilli 
ordinarily seen in sputum, and some are paler red, but all 
the forms occur. These bacilli may occur singly or in 
groups. The greater part of the sediment consists of undi- 
gested nuclei and a small amount of detritus. 

Animal inoculation gives the most satisfactory results 
if practised as follows : 

Take at least two centrifuge tubefuls of the fluid and cen- 
trifugalize for ten minutes. Decant the supernatant fluid 
and add about 10 m. of the original fluid to each tube. 
Stir up the sediments in the two tubes until thoroughly sus- 
pended ; then mix them and inoculate a guinea-pig subcu- 
taneously. Not over 30 m. of fluid should be used, since 
this is sufficient, and in most cases does not cause toxemia 
in the animal. 

Cultures from purely serous fluids are, in the vast majority 
of cases, sterile. In a few cases the writer has found the 
pneumococcus and streptococcus in pure culture. 

Ovarian and Parovarian Cysts. — The simple cysts of 
the ovary due to distention of Graafian follicles or to cystic 
change of corpora lutea, and the parovarian cysts contain a 
thin, clear, serous fluid of low specific gravity. 

The contents of the multilocular and papillary adeno-cys- 
tomata of the ovary are usually tenacious and mucous, of 
very varying specific gravity, from 1 005- 1 050, but usually 
between 1020 and 1024. The fluid generally contains much 
albumin and is rich in metalbumin, which is precipitated by 
alcohol, but not by acetic acid, nitric acid, or boiling, so that 
it can readily be distinguished from mucin. Before making 
the test the albumin must be removed. 

The cyst-contents are usually yellowish, but sometimes 
may be dark-red or chocolate-colored. Microscopically, 
red and white blood-globules, occasionally blood-pigment 
and cholesterin crystals, often fat-granules and large vacu- 



462 PATHOLOGICAL TECHNIQUE. 

olated cells, are found in the cyst fluid. Bizzozero considers 
cylindrical epithelial cells, ciliated and beaker cells, and col- 
loid concretions especially important from a diagnostic point 
of view. 

Pancreatic Cyst or Fistula. — The fluid obtained from 
a permanent fistula or large cyst of the pancreas contains 
much less solids than the normal pancreatic juice, and the 
trypsin ferment may be present in very small amount or 
possibly be entirely wanting. The fluid is colorless, alkaline 
in reaction, and has a specific gravity of about ion. It is 
characterized by three distinct properties on which its rec- 
ognition depends — namely : 

1. It splits up fat into fatty acids and glycerin. Mix to- 
gether equal parts of neutral olive oil and the alkaline fluid. 
Test with litmus-paper. Place the mixture in the incubator 
at 37 C, and test from time to time. If the fluid is pan- 
creatic, an acid reaction will be obtained in twelve to eighteen 
minutes. 

2. It transforms starch into sugar. Place in the incubator 
equal parts of a I per cent, aqueous solution of starch and 
of the fluid to be tested. In ten to twenty minutes test for 
sugar with Fehling's solution. 

3. It digests fibrin in an alkaline solution (trypsin ferment). 
Place some fibrin in the alkaline fluid and set it in the incu- 
bator. In one-half to one hour examine for peptones by 
the biuret test. Add caustic potash or soda and a few 
drops of a dilute solution of sulphate of copper. If pep- 
tones are present, a beautiful reddish-violet color will be 
produced. 

Dropsy of the Gall-bladder. — Puncture is generally 
not advisable. The fluid is usually colorless and mucoid or 
serous in character. All trace of biliary constituents may 
have disappeared. According to Lenhartz, numerous colon 
bacilli are usually present. 

Hydronephrosis and Renal Cysts. — The fluid is 
almost always clear as water, rarely reddish or yellow. 
Specific gravity always under 1020 (usually between 1010 
and 1015). Urea and uric acid are generally present, but 
may be absent. (Small amounts of urea are sometimes 



j 



CLINICAL PATHOLOGY. 463 

present in ovarian cysts. Albumin is slight in amount. 
Microscopically, almost nothing is found. 

Echinococcus Cysts.— The fluid is perfectly clear, free 
from albumin, and contains a little succinic acid and much 
chlorid of sodium. The specific gravity varies between 1008 
and 1013. 

Microscopically, often no traces of morphological elements 
can be found. Occasionally, however, hemosiderin granules 
or cholesterin crystals occur, or the characteristic structures 
from which a positive diagnosis can be made — namely, scoli- 
ces, hooklets, or pieces of cyst-membrane. 

A positive diagnosis from a chemical examination depends 
on showing — 

1. The absence of albumin. 

2. The presence of chlorid of sodium. 

Evaporate a drop of the fluid slowly on a slide, so as to 
get the characteristic crystals of chlorid of sodium. 

3. The presence of succinic acid. 

Acidify a little of the fluid with hydrochloric acid and 
evaporate to dryness. Extract the residue with ether. The 
crystallized material left on the evaporation of the ether, if 
dissolved in water, will give a rust-colored, gelatinous pre- 
cipitate with sesquichlorid of iron if succinic acid be present. 

Examination of the Sputum.— The secretion raised 
from the air-passages by coughing is almost invariably con- 
taminated with the secretion of the naso-pharynx and with 
particles of food from the mouth. In examinations of 
sputum these contaminations must always be borne in mind. 
The amount raised varies from a few c.c. to one or even 
several liters in twenty-four hours. 

The macroscopic appearances of the sputum depend on 
the varying proportions of mucus, pus, blood, and serum 
present. The tenacity is mainly due to the mucus. The 
reaction is usually alkaline. 

The general color, consistency, and separation into layers 
is best seen after the sputum has stood for some time in a 
tall glass. For more careful macroscopic examination small 
portions of the sputum are transferred to flat glass dishes, 
where they are spread out thinly by needles and examined 



464 PATHOLOGICAL TECHNIQUE. 

over black or white paper. Porcelain plates painted black or 
black paper itself can be used. The latter method is con- 
venient, because the sputum can be burned up with the paper. 

The constituents of the sputum which may be recognized 
macroscopically are few in number, and not so important as 
those which may be found microscopically. 

Macroscopic Examination.— 1. Caseous Masses. — In the 
sputum from tubercular cases small, opaque, yellowish-white 
masses from the size of a pin-head to that of a small pea 
can occasionally be found, which spread out beneath a cover- 
glass like a bit of cheese. They are small caseous masses 
which are valuable for microscopic examination because they 
usually contain tubercle bacilli and elastic fibers. 

2. Fibrinous casts of the bronchioles can usually be found 
in the sputum from the third to the seventh day in cases of 
acute lobar pneumonia. They appear as yellowish-white or 
reddish-yellow threads, 2 to 3 mm. thick and \ to several 
cm. long, and are often branched. The large ones are often 
rolled into balls, and show best after being shaken in water. 
Casts of the bronchi are found in cases of fibrinous bronchitis. 

3. Curschmann's spirals (Fig. 150) of twisted threads of 
mucus enclosing epithelial cells and leucocytes occur rarely, 
except in bronchial asthma. They appear macroscopically 
as grayish-white or whitish-yellow masses or threads, \to \\ 




Fig. 150. — Curschmann's spiral; X 425 (W. H. Smith; photo by L. S. Brown). 

mm. thick and \ to 8 cm. long, and often show a visible 
spiral arrangement. 

4. Dittrictis Plugs. — These are whitish-yellow masses from 



CLINICAL PATHOLOGY. 465 

the size of a pin-head to that of a bean, which are formed in 
cases of putrid bronchitis and of gangrene of the lung. 
They have a very fetid odor, a cheesy consistency, and are 
rather easily compressed. Besides organisms they contain 
numerous fat-crystals. 

-5. Shreds of tissue are found almost solely in gangrene of 
the lung, and are best recognized with the microscope. 

6. Concretions, portions of cysticercus membrane, etc., are 
rare in the secretion from the lungs. 

Microscopic Examination. — Microscopically, the sputum 
may show various kinds of cells, fragments of tissue, includ- 
ing elastic fibers, vegetable and animal parasites, and crystals. 

They will be taken up in order : 

1 . Red Blood-globules. — In fresh hemorrhages they appear 
normal, often in rouleaux. In old sputa many have lost 
their color. 

2. White blood-globules are almost invariably polynuclear, 
and the majority of them contain neutrophilic granules. In 
asthma, however, numerous eosinophilic and rather numer- 
ous basophilic leucocytes are regularly found. The leuco- 
cytes often contain pigment- or fat-granules. 

3. Epithelial Cells. — Pavement, cylindrical, and ciliated 
cells are found. The first come from the naso-pharynx ; the 
others usually from the trachea and bronchi, but may come 
from the nose. Desquamated alveolar epithelium is difficult 
to demonstrate. The pigmented cells found almost wholly 
in chronic passive congestion of the lungs are chiefly, 
perhaps entirely, desquamated alveolar epithelium. The 
pigment appears as yellowish, yellowish-red, or brownish- 
red granules or as yellow diffuse pigmentation. Occasion- 
ally, however, it surrounds granules of carbon, and then 
appears brownish or grayish-black. The pigment is derived 
from the blood, and will usually give the iron reaction (see 
page 185), but very young or old pigment will not. 

4. Fatty Detritus. — Fat-drops are frequently found, due to 
the fatty degeneration of cells. 

5. Elastic fibers (Fig. 151) occur singly, but more often as 
a network. They are recognized by their sharp, dark out- 

30 



466 



PA THOL GICA L TE CHNIQ UE. 



lines due to their high degree of refractiveness, and by their 
marked degree of resistance to acids and alkalies by which 
other like tissues, such as connective-tissue fibers, are de- 
stroyed. Elastic fibers are most abundant in the caseous 
masses above mentioned. When these masses cannot be 
found, the thicker portions of the sputum are squeezed be- 
tween a slide and cover-glass or between two slides, and ex- 
amined with a low power. The examination is rendered 
easier by mixing a little sputum with a 10 per cent, solution 
of caustic potash or soda. In certain cases it is necessary 
to mix together equal parts of the sputum and 10 per cent, 
caustic potash or soda, and to boil the mixture until the 
sputum is dissolved. The solution is then mixed with four 




Fig. 151. — Elastic fibers (after Striimpell). 

times its own volume of water and allowed to stand for 
twenty-four hours, when the sediment can be examined for 
the elastic fibers. 

Vegetable and Animal Parasites. — Of the vegetable 
parasites, the most important is the tubercle bacillus (for its 
examination see page 348). Other bacteria sometimes ex- 
amined for are the pneumococcus, the influenza bacillus, and 
actinomyces. 

W. H. Smith's Method of Staining Bacteria in Sputum. — 
This has been found particularly useful in demonstrating the 
pneumococcus in the sputum , The sputum or other material 
should be fresh. The cover-glasses should be spread as 
thinly as possible and fixed by passing three times through 
the flame in the usual manner. 

I. Stain in aniline-gentian- violet solution for a few 
seconds, gently warming until the staining fluid steams. 



CLINICAL PATHOLOGY. 467 

2. Wash in water. 

3. Cover with Gram's solution of iodin for thirty sec- 
onds. 

4. Wash with 95 per cent, alcohol until the color ceases 
to come out. 

5. Wash with ether for a few seconds. (To remove fat.) 

6. Wash in absolute alcohol for a few seconds. 

7. Stain one to two minutes in a saturated aqueous solu- 
tion of eosin. 

8. Wash with absolute alcohol for a few seconds. 

9. Clear with xylol. 
10. Mount in balsam. 

The pneumococcus is stained blue-black, while the capsule 
is stained pink. This method gives beautiful preparations. 
With the following modification it has been used by Smith as 
a routine stain for sputum. The advantage of this modifica- 
tion is that influenza bacilli and other bacteria which do not 
stain by Gram's method are clearly brought out, as are also 
eosinophilic leucocytes. This modification consists in wash- 
ing the preparation with Loffler's alkaline methylene-blue 
solution just after it has been stained with eosin, as described 
above, and then, after the excess of eosin has been removed 
by the methylene-blue, steaming the methylene-blue solu- 
tion for a few seconds while on the cover-glass. The prepa- 
ration is then washed in water, rinsed with alcohol, cleared 
with xylol, and mounted in balsam. 

Of the animal parasites, the entamoeba histolytica is some- 
times found secondaiy to an hepatic abscess which has perfo- 
rated into the lung (see page 381). Portions of the membrane 
of an echinococcus cyst or the hooklets from the head may be 
found in the sputum, but infection with this parasite is very 
rare in this country. 

Of the crystals which occur in sputa, the most important 
are the Charcot-Leyden crystals, found mainly in bronchial 
asthma, and the crystals of the fatty acids, of cholesterin, 
and of hematoidin. Tyrosin and leucin are much more 
rare. 

The Charcot-Leyden crystals are colorless, elongated 
octahedra of varying size, soluble with difficulty in cold 



468 



PA THOL O GICAL TE CHNIQ UE. 



water, insoluble in alcohol, ether, chloroform, and dilute 
saline solution. 

Hematoidin crystals occur as ruby-red rhombic plates or 
columns. 




Fig. 152. — Charcot-Leyden crystals and eosinophilic leukocytes in a smear prepara- 
tion of sputum from a case of asthma (W. H. Smith; photo by L. S. Brown). 




IlG 153. — -Crystals of cholesterin (after Striimpell). 

Cholesterin crystals (Fig. 153) occur as the well-known 
small and large rhombic plates. 



CLINICAL PATHOLOGY. 469 

The fatty-acid crystals occur as long, pointed needles, 
either singly or in groups. They are easily soluble in ether 
or hot alcohol, insoluble in water and acids. 

Examination of the Gastric Contents. — The micro- 
scopic examination of the contents of the stomach is much 
less important than the chemical. Fresh blood is easily 
recognized by the microscope. Disintegrated blood must 
be examined for chemically by the hemin test, as follows : 

Mix a little of the suspected material with a crystal or two 
of common salt, or place it on the thin layer of salt formed 
by slowly evaporating a small drop of normal salt solution 
on a slide. Cover with a cover-glass, and run in enough 
glacial acetic acid to fill up the space between slide and cover. 
Warm the slide over a flame for three-quarters to one 
minute until bubbles arise, adding more glacial acetic acid as 
evaporation takes place, until a faint reddish-brown tint 
appears. Then let the acetic acid evaporate entirely, and 
run glycerin in from the edge of the cover-glass. Micro- 
scopic examination will show dark-brown rhombic plates or 
columns of hemin if blood is present. 

Shreds of tissue or bits of mucous membrane are some- 
times found in the vomitus or removed by means of a 
stomach-tube. Examination of them in the fresh condition, 
or, more satisfactorily, in stained sections after hardening and 
imbedding, will sometimes give definite information in regard 
to the condition of the mucous membrane, or render pos- 
sible the diagnosis of a malignant growth. 

Examination for Free Hydrochloric Acid. — Of the fol- 
lowing tests, that with Congo-paper is the quickest and 
easiest, but shows only that a free acid is present. To prove 
that the free acid is hydrochloric acid the phloroglucin- 
vanillin test or one of the others is necessary. 

1. Congo-paper is turned blue by free acids only. Free 
hydrochloric acid turns it of a cornflower-blue, a tint obtained 
with lactic acid only when in much greater concentration 
than is ever present in the stomach. Congo-paper is used 
simply by dipping it into the stomach-contents, preferably 
after filtration. 



470 PATHOLOGICAL TECHNIQUE. 

2. Giinsburg's Test with Phloroglucin-vanillin. — The solu- 
tion consists of — 

Phloroglucin, 2 ; 

Vanillin, I ; 

Absolute alcohol, 30. 

Three or four drops of this solution are placed with an equal 
amount of the nitrate from the stomach-contents in a porce- 
lain dish and carefully heated over a small flame. Keep the 
dish in constant motion, and do not allow the mixture to 
boil, because boiling prevents the reaction from taking place. 
If free hydrochloric acid is present, a rose-red mirror is pro- 
duced. The phloroglucin-vanillin solution does not always 
keep well, so that it is best to keep alcoholic solutions of 
phloroglucin and of vanillin in separate bottles, and to mix 
together one or two drops of each when required. 

3. Boas' Resorcin Test. — The solution consists of — 

Resublimed resorcin, 5 ; 

Cane-sugar, 3 ; 

Alcohol, 94 per cent., ad 100. 

It is used in the same manner as the phloroglucin-vanillin 
test. A similar but more transient mirror is produced. 

Topfers Dimethyl-amido-azo~be?izol test is highly recom- 
mended by Simon as superior to the phloroglucin-vanillin 
test. "One or two drops of a 0.5 per cent, alcoholic solu- 
tion is added to a trace of the gastric contents, which need 
not be filtered; in the presence of free HC1 a beautiful 
cherry-red color develops, which varies in intensity according 
to the amount of free HC1 present." 

Examination of the Feces. — In examining for worms 
and their eggs it is often best to dilute the feces with water, 
and then to examine the sediment both macroscopically and 
under the microscope. (For Entamoebas see p. 381.) The 
other protozoa are best looked for in fresh slide preparations. 

For the cholera vibrio see p. 355, for the typhoid bacillus 
see p. 302. 

The membranous casts sometimes found in feces consist 



CLINICAL PATHOLOGY. 47 1 

almost wholly of mucus, cylindrical, epithelial cells, and 
leukocytes. Bits may be examined fresh, or the casts may 
be hardened and sections made and stained after embedding 
in celloidin. 

Bass recommends that feces which have been made 
fluid with water be centrifuged and the supernatant fluid 
containing vegetable debris be poured off. The sediment 
contains the hookworm eggs if present. Pour on the sedi- 
ment a calcium chloride solution of sp. gr. 1050. Again 
centrifuge and decant. Next add calcium chloride solution 
of a sp. gr. 1250 and centrifuge. This brings to the surface 
the hookworm eggs, which may be pipetted off. As a rule, 
the finding of hookworm eggs is very easy without such a 
technic. The eggs of Trichostrongylus greatly resemble 
those of hookworm, but are larger — 73 to 91 \x long. In per- 
fectly fresh feces Strongyloides are present as worm-like 
embryos, while hookworm gives only two to four segment 
eggs. 

Examination of the Urine. — Only those points are 
mentioned which come within the province of the patholo- 
gist. 

Of the animal parasites, the bilharzia, the hookworm, the 
echinococcus, and the filaria sanguinis hominis are the only 
important ones (see pp.405, 406). 

Of the vegetable parasites, tubercle bacilli and gonococci 
are the most common ; actinomycetes are very rare. 

New growths in the kidneys are accompanied with hemor- 
rhage in less than half of the cases, while new. growths in 
the bladder almost invariably give rise to it. Fragments from 
new growths in the bladder are rare. A diagnosis of malig- 
nant disease from cells only is impossible. Pieces of tissue 
which show on microscopic examination the characteristic 
structure of cancer or other neoplasm must be obtained ir? 
order to render a diagnosis possible from the pathological 
(but not from the clinical) standpoint. 



POST-MORTEM EXAMINATIONS. 



Introduction. — The method of making post-mortem ex- 
aminations most generally followed in this country and 
abroad is that originally taught by Virchow. It has been 
variously modified in minor details by his pupils and fol- 
lowers. The strongest adverse criticism which can be made 
of the method is that it works best when the various organs 
in the body are nearest normal. Its chief fault lies in the 
early separation of the different structures from each other, 
so that interesting pathologic relations are often overlooked 
and destroyed. Special procedures are advised for certain 
conditions, but emphasis is not placed on them. 

Two other methods of making post-mortem examinations 
were developed besides that of Virchow's, but have never 
attained the same general recognition, although both contain 
principles of great value. 

The technique of Rokitansky, lately best exemplified by 
Chiari, appeared in printed form a number of years ago. Its 
fundamental principle is to examine and open every organ in 
situ before removing it, so that all abnormal relations between 
orgrans, blood-vessels, and other structures can be discovered 
and preserved intact. 

Recently, Hauser has published Zenker's post-mortem 
technique. Its main principle is to remove the organs in 
block, so that they can be examined from every side and in- 
cised before they are separated from each other. 

In both methods the incisions in the various organs, and 
especially the heart, differ more or less radically from those 
recommended by Virchow. 

A knowledge of all three forms of procedure is useful. It 
broadens one's point of view, and tends to keep one's mind 
open to possibilities. A list of the best publications on post- 
mortem technique is appended at the end of this section of 
the book. 

472 



POST-MORTEM EXAMINATIONS. 4?$ 

The problem offered by an autopsy is often solved in part 
or wholly by the macroscopic post-mortem examination. 
More frequently, however, the complete and final solution 
is reached only after careful bacteriological and histological 
study. The post-mortem examination may, therefore, be 
looked upon as the beginning of the solution of the prob- 
lem. Its particular function is to demonstrate in the indi- 
vidual case all congenital or acquired abnormalities, all 
macroscopic lesions, and to explain all gross mechanical 
questions. It furnishes the material for bacteriological and 
histological study. Perfectly to accomplish its purpose a 
post-mortem examination must be made in a careful, sys- 
tematic manner. 

While a general method of procedure is advisable, it will 
often be found advantageous, or even necessary, to depart 
from it. According to Orth, "the chief requisite of every 
exact post-mortem examination is this, that no part shall be 
displaced from its position until its relations to the surround- 
ing parts are established, and that no part shall be taken out 
by whose removal the further examination of other parts is 
affected." 

The order and method of procedure in making a post- 
mortem examination, including the various incisions, may be 
said to have been planned for the routine examination of 
normal or diffusely diseased organs. As soon as a notice- 
able focal lesion is present the order of procedure and the 
customary method of removal and of incision must be so 
altered as best to display the lesion. 

Instruments. — The following instruments will be found 
extremely useful in the autopsy-room, although not all of 
them are necessary: 

The autopsy-table should be large, so as to accommodate 
on it the instruments and several dishes in addition to the 
body. It should have a slightly raised edge, and should 
slope gently toward an opening in the center for the escape 
of fluids. The table is best made of zinc, and along one 
edge should have a centimeter scale. The water for use on 
the table is best supplied by a rubber tube from an overhead 
pipe reaching to within 60 to 100 cm. of the table. 



474 



PA THOL O GICAL TE CHNIQ UE. 



The scales for weighing the various organs should have a 
large pan and gram and kilogram weights. 







Fig. 154. — Instruments for use in the autopsy-room: a, Saw; b, holder for the 
head; c, steel hammer with wedge end and blunt hook on the handle; d, costo- 
tome; e, bone-cutter; /, hatchet-chisel; g, autopsy-knife. 

A band-saw will be found very useful for sawing bones for 
the inspection of the marrow, and for calcified and osseous 
tumors. 

The best autopsy-knife is a stout, broad-bladed knife with 



POST-MORTEM EXAMINATIONS. 475 

bellied edge and heavy handle. The blade should measure 
about 12 cm. in length and 3 cm. in width ; the handle should 
be 12 cm. in length. Many operators prefer a somewhat 
smaller knife than this. 




Fig. 155. — Myelotome. 



Amputating-knives of different sizes are useful for long, 
deep cuts into organs and tumors. 

A myelotome is a short, thin, narrow knife-blade, 1.4 cm. 
long and 4 mm. wide, set obliquely on a slender steel stalk 




Fig. 156. — Enterotome. 



ending in a wooden handle (Fig. 155). It is used only for 
cutting the cord squarely across in removing the brain. 

Cartilage-knives and scalpels of different sizes are useful 
for a variety of purposes.' 




Fig. 157. — Luer's double rachiotome. 

Scissors, both straight and curved, should be of various 
sizes. A medium-sized and a fine pair should each have 
one probe-pointed blade. 

An enterotome is a long, straight pair of scissors, of which 



476 PATHOLOGICAL TECHNIQUE. 

one blade is longer than the other and blunt at the extrem- 
ity (Fig. 156). A hook at the end is not advisable. The 
instrument is used in opening the heart and the intestines. 

A saw with movable back and rounded end will be found 
the most generally useful for opening the skull and the 
spinal canal. An ordinary meat-saw is preferred by some, 
but cannot be used on the vertebrae. 

Luer's double rachiotome, or adjustable double saw (Fig. 
157), is very useful in removing the cord, and is the safest 
instrument to put into the hands of beginners. 

Forceps : several sizes, large and small, mouse-toothed. 

Costotome : heavy bone-shears for cutting the ribs. 

A powerful bone-cutter, with short blades, 5 cm. long, set 
at an angle of about 45 ° to the handles, which are 36 cm. 
in length, is employed for dividing the arches of the cervical 
vertebrae and for other purposes where ordinary bone-cut- 
ters will not do. 

A chisel with 2 cm. cutting edge, for exposing the marrow of 
the long bones, removing portions of the base of the skull, etc. 

A hatchet-chisel of steel for starting the calvaria and spinous 
processes after sawing the skull and the vertebral column. 

Soft-iron hammer with wooden handle. 

Steel hammer with wedge end, and blunt hook on the 
handle. 

Holder for the head while sawing the skull. 

Autopsy-needles, long and a little curved. 

Probes of flexible metal ; also fine glass probes for small 
blood-vessels or ducts. 

Grooved director. 

Pans for holding water, organs, etc. 

Boards, square or oblong, 30 X 30 or 30 X 50 cm., on 
which to lay instruments or cut organs. 

Sponges. 

Catheters. 

Strong hemp twine is the best for sewing up the body. 

Glass graduates for measuring fluids. 

A block of wood with shallow depression for the neck ; for 
use while opening the head. 
Vise. 



POST-MORTEM EXAMINATIONS. 4/7 

Small cup or dish for removing fluid from cavities. 

General Rules. — The room for an autopsy should be 
well lighted, otherwise the finer changes in the tissues cannot 
be recognized. Artificial illumination is not good, because 
the colors of the tissues are entirely changed by the yellow- 
ness of the light. 

Before beginning an autopsy the necessary instruments 
should be arranged on a short board on the autopsy-table in 
the order in which they are most likely to be used. 

The operator stands on the right side of the body. This 
position he rarely leaves except for some definite purpose; 
for example, in opening the skull he stands at the head. 

Order and cleanliness are the first points to be insisted 
upon at every autopsy. Clean water should always be at 
hand for washing the instruments and for keeping the hands 
free from blood and pus. The cut surface of an organ should 
not be washed with water except to remove blood; gently 
scrape the surface with the knife held obliquely. 

In cutting, the knife should be drawn, not pressed or 
shoved into the tissues. According to Virchow, a broad, 
clean cut into an organ, even if incorrectly made, is much 
better than several short cuts which leave a ragged surface. 

The autopsy-knife should be grasped in the hand as if to 
cut bread. In using this knife the main movement should be 
from the shoulder, not from the wrist as in dissecting. It 
goes without saying that the sharper the knife the better. 

In cutting the brain and cord, especially if their consistency 
is lessened, moisten the knife to prevent the tissue from stick- 
ing and tearing. 

Before beginning an autopsy it is important to know the 
main points in the clinical history of the case, as they may 
greatly lighten the work of investigation by calling attention 
to those organs that require special examination. 

The record of an autopsy should be dictated by the ope- 
rator as he proceeds with the examinaticn of the case, and 
should be as nearly as possible an objective description of 
the appearances found. Only the anatomical diagnosis 
should express the opinion of the operator. If it is not 
convenient to dictate the autopsy during its performance, 



47^ PATHOLOGICAL TECHNIQUE. 

the description of the lesions certainly ought to be made 
with the organs in sight, and not from memory after the 
lapse of hours or even days, when many of the details may 
be forgotten. Later, the results of the bacteriological and 
histological examinations should be added to the autopsy 
report, so as to make the case complete. 

The thin rubber gloves now used by surgeons are very 
useful in making post-mortem examinations, especially in 
septic cases and while opening the stomach and intestines. 
Rubber cots for the fingers are often useful. 

For cuts on the fingers use celloidin dissolved in equal 
parts of alcohol and ether, instead of flexible collodion, be- 
cause the latter will not stick. A cut received during an 
autopsy should immediately be washed thoroughly. For 
protection during the rest of the autopsy, use a rubber glove 
or cover the cut with celloidin. 

After an autopsy the operator should scrub his hands 
thoroughly with soap and brush, just as a surgeon does be- 
fore an operation, and then use, if he so desires, an antiseptic 
solution, such as corrosive sublimate (i : 2000) or 70 per cent, 
alcohol. For removing odors from the hands, turpentine will 
often be found serviceable, or a saturated solution of per- 
manganate of potassium followed by oxalic acid. 

For infections of slight wounds, such as scratches, or such 
as occur in hair-follicles, the best treatment within the first 
twenty-four hours is to bore into them with a sharp-pointed 
orange-wood stick dipped in strong carbolic acid, followed by 
washing with 95 per cent, alcohol. The procedure is prac- 
tically painless, and the infection is stopped in the very be- 
ginning. Where the infection has spread, surgical treatment 
must be resorted to. 

Suggestions to Beginners. — In a case of general 
miliary tuberculosis the older focus from which the organ- 
isms have spread must always be found. Look especially 
for tubercular thrombi in the pulmonary veins as a frequent 
source of the general infection. 

In a case of embolism hunt for the thrombus, bearing in 
mind, however, that the whole of a thrombus may become 
free and form an embolus. An arterial embolus may be due 



POST-MORTEM EXAMINATIONS. 479 

to a venous thrombus, in which case it must have passed 
through an open foramen ovale, except in the case of 
thrombi of the pulmonary veins. 

In acute peritonitis always seek for a source of infection 
(appendix, female genitals, gastro-intestinal tract, etc.). It 
cannot always be found. 

In hemorrhage from the stomach associated with cirrhosis 
of the liver look for rupture of dilated esophageal veins. 

In cases of more or less sudden death, especially if pre- 
ceded by signs of asphyxia, always examine the pulmonary 
artery in situ for possible emboli. In cases of instantaneous 
death examine the coronary arteries. 

Private autopsies must often be made under many dis- 
advantages, and, when out of town, not infrequently in a 
short space of time. It is always important to warn the 
attending physician not to allow the undertaker to inject the 
body before the autopsy, because the color and consistency 
of the organs are so changed by most injecting fluids that it 
is difficult to recognize the pathological processes. If there is 
danger of post-mortem changes, have the body packed in ice. 

A regular autopsy-bag will be found very convenient for 
carrying to private autopsies. It is made of leather lined 
with rubber, and measures about 40 X 18 X 20 cm. Loose 
within it is carried a rubber bag 40 X 24 X 20 cm., shaped 
like a short envelope with a flap (22 cm. long) on one side, 
for bringing away any organs that demand further examina- 
tion. The case of instruments should contain one or two 
autopsy-knives, two scalpels, a pair of forceps, one or two 
pairs of scissors, an enterotome, a steel hammer with wedge- 
end and with a blunt hook on the handle, a small chisel, a 
saw with detachable handle and back, an autopsy-needle, 
and a probe ; free within the bag should be carried a spool 
of strong twine, a costotome, a long slender knife for use in 
removing the brain, a hammer with soft iron head, and a 
sponge. In rare cases additional instruments may be re- 
quired. A white duck apron for personal use will always 
be found convenient. It is also well to carry along several 
blood-serum tubes and a platinum needle for making cultures 
at the autopsy. When there is a lesion of the nervous sys- 



480 PATHOLOGICAL TECHNIQUE. 

tern it is advisable to bring a jar of a 4 per cent, solution of 
formaldehyde and to place the tissue in the fluid at the 
autopsy, as otherwise it is not easily gotten to the laboratory 
in good condition. 

At the house can always be obtained a slop-pail, a wash- 
bowl, a pitcher of water, several newspapers, and an old 
sheet. The body is usually on an undertaker's frame, but 
it may be in an ice-box or on the bed. The examination of 
the chest and abdomen can be made in any of these posi- 
tions. If, however, the body is in an ice-box, it must be 
raised to the level of the top of the box in case it is neces- 
sary to open the head. 

The clothing on the body can be removed, or, if only a 
shirt or a night-dress, is best slit down the middle and turned 
out over the arms. Tear the sheet into four equal pieces. 
Fold and tuck in one piece on each side of the trunk and 
neck, allowing the outer portion to fall over the arms. Fold 
and lay the third piece on the lower extremities, tucking the 
upper end beneath the clothing below the pubes. The 
fourth piece can be placed beneath the head if it is to be 
opened. This procedure leaves the front of the thorax and 
abdomen free for operation and protects the rest of the body 
and the clothing. On the thighs place one or two folded 
newspapers, and on these the necessary instruments. On the 
legs place the bowl containing only a dampened sponge. 
If the undertaker has not put a rubber sheet on the floor 
beneath the body and on the side where the operator is to 
stand, newspapers should be spread to protect the carpet. 
Place the slop-pail on the rubber sheet within convenient 
reach. Having thus made all arrangements, even to the 
threading of his needle, the operator is ready to begin. 

If the cord and brain have to be examined as well as the 
body, it is best to do the cord first, so as to avoid the leak- 
age that might otherwise occur from the trunk-cavities if 
they had been opened first. To support the head while 
opening it, use a stick of wood, a brick, or, in case of neces- 
sity, the instrument-box wrapped in a newspaper. 

At a private autopsy cleanliness is extremely important. 
If there is no ' undertaker or nurse present, the operator 



POST-MORTEM EXAMINATIONS. 48 1 

himself must see that everything is cleaned and put in order 
before leaving, that all the blood-stains are removed from the 
dishes, and that all papers and soiled cloths are burned or 
rolled up and left in a neat bundle for the undertaker to dis- 
pose of. Ground coffee thrown on a shovelful of burning 
coals will be found helpful in disguising the odor in the 
room after an autopsy. 

EXTERNAL EXAMINATION OF THE BODY. 

External examination is often of great importance, espe- 
cially in medico-legal autopsies, and should never be ne- 
glected, as it may throw great light on lesions found within 
the body. It should be systematic and careful, and is best 
taken up in the following order: 

I. Inspection of the Body as a Whole. 

1. Sex. 

2. Age. 

3. The body-length should be measured on the table beside 
the body, between points opposite the vertex of the head 
and the sole of the foot beneath the ankle. 

4. The development of the skeleton has reference to the bony 
framework, which may be powerful, slender, or deformed. 

5. The general nutrition is shown by the amount of mus- 
cular development and of subcutaneous fat-tissue. The 
latter is judged by pinching up folds of skin. 

6. The general condition of the skin includes amount of 
elasticity, bronzing, jaundice, edema, and decubitus. 

7. Post-mortem discolorations may be divided into three 
varieties : 

(a) Hypostasis of blood, or the settling of blood into the 
lowest lying blood-vessels ; this form of discoloration dis- 
appears on pressure. 

(b) Diffusion of blood-coloring matter out of the vessels into 
the surrounding tissues (due to blood-pigment being set free 
by post-mortem decomposition) ; does not disappear on 
pressure. 

(c) The greenish discoloration, usually seen earliest over 
the abdomen, is due to sulphide of iron formed through 
decomposition of the tissues. This discoloration is import- 

31 



482 PATHOLOGICAL TECHNIQUE. 

ant, as it may modify the interpretation of appearances 
observed in the internal organs. 

8. Post-mortem rigidity, degree and extent. It begins in 
the maxillary muscles, and spreads gradually from above 
downward, disappearing later in the same order. It is most 
marked, and lasts longest in muscular individuals who have 
been ill but a short time. Cholera furnishes the most marked 
cases. The rigor disappears quickest in cachectic diseases. 
When once it has been forcibly overcome, it does not recur. 
The time of beginning after death varies widely — from ten 
minutes to seven hours. 

II. Special Inspection of the Different Parts of the Body 
The examination should begin with the head. Any lesion 
or abnormality found should be carefully noted. Particular 
attention should be paid to the condition of the pupils and to 
the color of the sclera. Then follow in order the neck, the 
thorax (size and shape), the abdomen (distended or retracted),, 
the genitals, and the extremities. 

INTERNAL EXAMINATION OF THE BODY. 

The opening of the body-cavities is described first, be- 
cause the brain is relatively much less frequently the seat of 
disease, and because in this country it is often impossible to 
obtain permission to open the head. Moreover, the lesions 
in the body often throw much light on those to be expected 
in the brain. The advantage of examining the brain first, 
particularly in those cases in which the important lesions 
are cerebral, is said to be that the amount of blood in the 
cerebral vessels can be more accurately determined. After 
the heart has been removed some of the blood in the brain 
may escape through the severed vessels below. 

In routine examinations, however, the body is usually 
examined first, then the brain, and finally the cord. It is 
not a bad practice to remove the calvarium, to examine the 
meninges over the upper surface of the cerebrum, and then 
to make the examination of the body before removing the 
brain. In this way any change in the blood-supply of the 
cerebral vessels would be observed. 



POST-MO R TEM EXAMINA TIONS. 



483 



Opening of the Abdominal Cavity. — In the exam- 
ination of the body the peritoneal cavity is opened first, the 
two pleural cavities next, and the pericardial cavity last. The 
cavities and their contents are to be inspected in the order 
and at the time that each is opened, but the organs are to 
be removed from the cavities for further examination in the 
reverse order, beginning with the heart. 

The primary or long anterior incision to bare the thorax and 
to open the abdomen (Fig. 158) should extend from the larynx 
to the pubes, passing to the left of the umbilicus, so as not 
to cut the round ligament. In cutting, the handle of the 
knife is depressed so as to use the belly of the blade rather 




Fig. 158. — Primary incision in the body (Nauwerck). 



than the point. An incision beginning as high as the chin 
is, unfortunately, rarely allowable. Over the sternum the 
cut should extend down to the bone; over the abdomen, 
however, only into the muscles, or in fat people through the 



4&4 PATHOLOGICAL TECHNIQUE. 

muscles into the subperitoneal fat-tissue. To open the ab- 
dominal cavity, nick carefully through the peritoneum just 
below the sternum, introduce the first and second fingers of 
the left hand, and while making strong upward and outward 
traction on the right abdominal flap extend the incision to 
the pubes. Some operators prefer to separate the fingers 
and to cut between them. 

The abdominal flaps are rendered much less tense by cut- 
ting the pyramidales and recti muscles from below just above 
the pubis. Care must be taken not to injure the overlying 
skin. The abdominal cavity can now be examined, but 
more room will be obtained if the skin and the underlying 
muscles be first stripped back from the thorax to about 5 
cm. outside of the costochondral line. 

The operation is most easily and neatly done by lifting the 
skin directly away from the chest-wall or turning it forcibly 
out with the left hand, and then* cutting the tense tissue close 
to the cartilages and ribs with long sweeps of the knife held 
almost flat. The operation begins over the lower border of 
the ribs and extends upward. In dissecting off the skin and 
muscles from the left side the right hand works underneath 
the left. The mammae can easily be incised from the under 
side of the flap, and if necessary the axillary lymph-nodes 
can be reached by dissecting the skin farther out, especially 
over the clavicle. Before beginning the inspection of the 
peritoneal cavity it is important to examine first the surface 
of the incision into the abdomen, noting the thickness and 
color of the fat-tissue and the condition of the muscles. 

Inspection of the Abdominal Cavity. — The character 
of any fluid present should be determined and its amount 
measured or estimated. The simplest way to remove it is to 
dip it up with a small cup or dish and pour it into a glass 
graduate for inspection and measurement. If the presence 
of gas within the peritoneal cavity is suspected, a small 
pouch should be formed in the first incision as soon as it has 
been made and water poured in. The first opening into the 
'abdominal cavity should then be made with the point of a 
scalpel at the bottom of the water, through which the gas, 
if present, will escape in bubbles. 



POST-MORTEM EXAMINATIONS. 4§5 

The various abdominal organs and their relations to eacji 
other are to be investigated in situ by sight and by touch, 
As a rule, examine first the gastro-intestinal tract, including 
the appendix and the mesenteric lymph-nodes. Ulcerations 
of the intestine can often readily be made out through the 
walls. The examination of the spleen, liver, kidneys, and 
pelvic organs follows. The pancreas is easily reached by 
tearing through the omentum between the stomach and the 
colon, so as to open the lesser peritoneal cavity. 

After the inspection of the abdominal organs the position 
of the diaphragm is to be ascertained on both sides in the 
costochondral line by measuring with the right hand passeql 
palm upward underneath the ribs, and the left hand outside 
at the corresponding height to mark the position of ribs or 
intercostal spaces. On the right side the hand is to be 
passed up on the outside of the falciform ligament. Nor- 
mally, the diaphragm stands at the fifth rib on the left side, 
and at the fourth rib or fourth interspace on the right. 

Opening of the Thorax. — To open the thorax, cut 
through the cartilages close to the ribs from the second 
down (about 5 mm. distant) with a scalpel held nearly hori^ 
zontal, so that as one cartilage is cut through the handle of 
the scalpel will strike the next below and prevent the blade 
from penetrating too far and injuring the lung. In young 
people the cartilages can be cut easily by one long stroke on 
each side, but care must be taken not to go too deep. If 
the intercostal muscles are not divided by the same opera- 
tion, the sternum can be depressed by the left hand and the 
muscles severed by one pass of the knife on each side. The 
lower end of the sternum can now be elevated and freed 
from below upward from the diaphragm and pericardium 
until the first rib is reached. The cartilage of this rib is to 
be cut about 1 cm. farther out than the others, and from 
below upward toward the clavicle, with the handle of the 
knife beneath the elevated sternum and with the point and 
edge of the knife directed upward and a little outward. The 
sternum is then to be still further freed from the anterior 
mediastinal tissue until its upper end is reached. The sterno- 
clavicular joint on the left side can now be easily opened 



486 PATHOLOGICAL TECHNIQUE. 

from below by entering a scalpel just above the cartilage of 
the first rib, and following the irregular line of the joint 
around the end of the clavicle, while at the same time draw- 
ing the sternum over to the right side of the body. The 
right sterno-clavicular articulation is to be opened by con- 
tinuing the incision of the scalpel over the upper end of the 
sternum and into the second joint. The advantage of this 
method is that there is much less danger of wounding the large 
vessels at the base of the neck, and thus of mingling blood 
with any exudation which may happen to be present in the 
pleural cavities. If preferred, however, the articulations can 
be opened and the cartilages of the first ribs cut from above 
before freeing the sternum from the diaphragm. In this case 
enter a short, sharp, narrow-bladed scalpel held vertically, 
but loosely, into the left joint on its upper side, starting the 
incision just outside of the attachment of the sternal end of 
the sterno-mastoid muscle, and cut around the end of the 
clavicle by a series of short up-and-down strokes, allowing 
the blade to follow the irregular line of the joint. After cut- 
ting through the joint continue the incision outward and cut 
through the cartilage of the first rib. 

If the cartilages are calcified, use the costotome and cut 
through the ribs, as more room can be gained in this way, 
and they are more easily cut than calcified cartilages. When 
for any reason it is not permitted to open the thorax, the 
organs within it can be obtained through the opening into 
the abdominal cavity by freeing the diaphragm from the ribs, 
and removing first the heart and then the lungs. The 
sternum should be inspected at the time of its removal. It 
is perhaps best to examine next, especially in children, the 
epiphyses of the ribs at the costochondral line for any evi- 
dence of thickening. 

Inspection of the Pleural Cavities. — In the pleural 
cavities, as in the peritoneal cavity, the character and amount 
of any abnormal contents must be determined. If, from the 
clinical history or from any other reason, the presence of 
air in a pleural cavity is suspected, a pouch should be formed 
over the ribs by aid of the skin-flap and filled with water. 
The pleural cavity is then to be pierced with a scalpel 



POST-MORTEM EXAMINATIONS. 487 

through the bottom of the pouch. Air, if present, will 
bubble up through the water. 

Slight adhesions are best torn through or cut. If the 
lungs are firmly attached, it is best to strip off the costal 
layer of the pleura with the lung. This is most easily done 
by starting the anterior edge of the costal pleura with the 
handle of the scalpel, and working in first a finger and then 
the whole hand until the pleura is entirely free. In passing 
the hand into the pleural cavities protect the back of it, 
especially if the ribs have been cut through, by folding the 
skin-flap in over the edge of the ribs. 

If desired, the lungs can be drawn forward, examined over 
their whole extent, even incised, and then replaced until the 
heart has been removed. In the connective tissue of the 
anterior mediastinum there is almost always a certain amount 
of emphysema due to the removal of the sternum. Emphys- 
ema due to laceration of lung-tissue is more marked in the 
upper half of the mediastinum, and usually extends up into 
the neck. The thymus gland attains its full development at 
the end of the second year, after which time it usually 
gradually disappears. 

Opening of the Pericardium. — To open the pericar- 
dium, seize the sac near the middle with fingers or forceps, 
snip through the wall with knife or scissors, and with either 
instrument cut upward to where the pericardium is reflected 
over the large vessels, downward to the lower right border, 
and lastly to the apex. By gently raising the apex of the 
heart the amount of fluid in the pericardial cavity can be 
seen. The normal amount is about a teaspoonful, but it may 
be increased to 100 c.c. in cases where the death-agony is 
prolonged. Pericardial adhesions should be broken through 
with the fingers. If this is impossible, the heart must be 
incised through the pericardium. 

External Inspection of the Heart. — Determine first 
the position, size, and shape of the heart, and the degree of 
distention of the different parts. The right ventricle and 
both auricles are usually distended with blood, which may 
be fluid as in death from suffocation or more or less coagu- 



PA THOL O GICAL TE CHNIQ UE. 

lated. The left ventricle is contracted and empty unless the 
individual has died from paralysis of this part of the heart, 
when it will be found distended with blood (condition of 
greatest diastole). 

Opening: of the Heart. — The heart may be opened in 
situ or after removal from the body. Except in certain cases, 
to be spoken of later, it usually will be found advisable to 
remove the heart before making any incision into it, for the 
reason that it can be more perfectly opened after removal, 
especially by beginners, and the danger of contaminating 
any bacterial lesions of the valves is lessened. 

To remove the heart, grasp it gently near the apex with 
the left hand, supporting it further, if necessary, by one or 
two fingers placed above the coronal suture, and lift the 
whole heart vertically upward. Then cut its vessels from 
below upward with the knife held transverse and oblique. 
Divide in turn the inferior vena cava, the pulmonary veins on 
both sides, the superior vena cava, the pulmonary artery, 
and the aorta. Go deep enough to remove the auricles 
entire, but avoid injury to the underlying esophagus. 

For making the incisions to open the heart either a long, 
slender-bladed knife or long, straight scissors may be used. 
The heart should be placed on a board with its anterior sur- 
face up. The right auricle is opened by cutting from the 
orifice of the inferior vena cava into that of the superior, 
and from the latter into the auricular appendage. The first 
incision to open the right ventricle is made through the tri- 
cuspid valve and the wall of the ventricle along the under 
surface of the right border of the heart. It should be 
carried to the end of the ventricle, which does not reach 
quite to the apex of the heart. The second incision begins 
about the middle of the first, just above the insertion of the 
anterior papillary muscle (which should not be cut), and is 
carried through the pulmonary valve well over on the left 
side along the left border of a narrow, projecting ridge of 
fat-tissue usually present, so as to pass between the left ante- 
rior and the posterior segments of the valve. 

The left auricle is opened in a manner similar to the right 



POST-MORTEM EXAMINATIONS. 



489 



by incisions joining the four orifices of the pulmonary veins 
and extending into the auricular appendage. 

The first incision into the left ventricle is through the 
mitral valve along the left border of the heart (i. e. the 
middle of the external wall of the left ventricle), between 
the two bundles of papillary muscles, to the apex of the 
heart The second incision begins at the termination of the 
first at the apex, and is carried up close to the interven- 
tricular septum, parallel to the descending branch of the 




Fig. 159. — Heart, showing incisions. 

anterior coronary artery and about 1 cm. from it. The 
upper portion of the incision should pass midway between 
the pulmonary valve and the left auricular appendage. 
Ordinarily, one of the aortic cusps is divided, but this may 
be avoided, if desired, by dissecting away to some extent the 
pulmonary artery from the aorta and carrying the incision 
well over to the right between the right posterior and ante- 
rior valve-segments. As each auricle is opened the blood 
and clots it contains should be carefully removed and the 
auriculo-ventricular valves carefully inspected from above. 
In certain cases — as, for instance, extreme stenosis — it may 
be preferable not to cut through the valve, but to begin the 



/ 



490 PATHOLOGICAL TECHNIQUE. 

incision in the ventricular wall below the valve. The ven- 
tricular cavities should in like manner be freed from clots 
and the valves closely inspected. The coronary arteries 
should always be opened by means of small, narrow-bladed, 
probe-pointed scissors as far as they can be followed. The 
examination of the descending branch of the anterior artery 
is especially important. The posterior coronary is best 
opened by placing the tip of the left fore finger in the aorta 
over the orifice of the artery, and cutting from without in 
toward the finger-tip until the vessel is reached, when it can 
easily be slit up. In this way injury to the aorta is avoided. 

In cases of more or less sudden death with symptoms of 
asphyxia the pulmonary artery should always be opened in 
situ before removal of the heart, in order to examine for pos- 
sible emboli, because they often lodge just at the point where 
the vessels are severed in removing the heart and lungs, and 
easily may slip out unobserved. The simplest operation is 
to thrust a sharp-pointed scalpel through the artery just 
above the valve on the left side in the line of incision already 
described, and to cut upward until the branches to the right 
and left lungs are reached. If desired, this incision may be 
extended down through the pulmonary valve and the ven- 
tricular wall along the line given for the second incision in 
the right ventricle. 

The water-test for the competence of the valves of the 
heart is not very reliable, especially for the auriculo-ven- 
tricular valves, and is not so much used as formerly. 
Inspection and measurement of the valve after the heart 
has been opened will usually enable one to judge fairly 
accurately concerning the degree of competence. Before 
applying the test to the aortic valve the first incision into 
the left ventricle must be made and the cavity freed from 
clots, so that no obstruction will exist below the valve. 
Then the heart is to be held so that the aortic valve is per- 
fectly horizontal, and water poured in from above to float the 
cusps out. If competent, they should keep the water from 
flowing through. If, however, in holding the heart the nor- 
mal relations of the valve and the surrounding parts are not 



POST-MORTEM EXAMINATIONS. 491 

maintained, the valve may leak. A second source of error 
is that the water may escape through the coronary arteries, 
branches of which have been cut in opening the ventricle. 
In testing the mitral valve the left auricle is first opened and 
the clots removed, so as to expose the upper surface of the 
valve. Then the nozzle of a syringe is introduced through 
the aortic valve and water forced in so as to float the mitral 
curtains up. The test, however, is very unreliable, because 
the parts cannot be placed under natural conditions. 

The pulmonary and tricuspid valves can, of course, be 
tested by methods similar to those already described. 

Increase or diminution in the size of the heart is best 
determined by weighing the organ after the removal of the 
clots. In certain cases, however, and in special investigations 
measurements of different parts of the heart are desirable. 
Roughly, the heart is the size of the individual's fist. 

The following weights and measurements are taken from 
Nauwerck 's Sectionstechnik : 

Weight of the heart averages in men, 300 gr. \ s\ f j. 

" " " " women, 250 " > 

Krause gives the average weight of the heart as 292 gr. 
Relative weight of heart to body in men, 1-169 1 g. 

" " " " women, I— 162 J 

Length of heart in men, 8.5-9 cm « \ /?• 

" " women, 8.0-8.5 " J 

Circumference of heart at base of ventricles, 28.8 cm. (Sappey). 

Thickness of wall of left ventricle, 1.1-1.4 cm. ) ~ 

^ V Krause. 

" " right " 0.5-0.7 " J 

Thickness of wall of left ventricle (without trabecule), 7-10 mm. "> n , 
<« « right « « « 2-3 " / 

Circumference of mitral orifice, 10.4 (W.), 10.9 (M.) ~] 
" tricuspid " 12.0 (W.), 12.7 (M.) ( 

« aortic " 7.7 (W.), 8.0 (M.) f rame ' 

" pulmonary orifice, 8.9 (W.), 9.2 (M.) J 
" ascending aorta, 7.4 cm. 
" pulmonary artery, 8.0 cm. [Buhl). 

The directions given for the removal and opening of the 
heart apply only when the organ is normal or contains 
lesions within itself which are not in continuity with any of 
the vessels entering into it. In aneurysm of the ascending 



49 2 PATHOLOGICAL TECHNIQUE. 

aorta, in thrombosis of a vena cava, and in a number of dif- 
ferent lesions connected with the heart or with the vessels 
given off from it, it is important to examine these vessels 
and to open them while they are still in continuity with the 
heart. For this purpose it is often necessary or advantageous 
to remove the thoracic organs in one piece, so as to be able 
to examine the central circulatory apparatus in continuity 
from the front and back before disturbing any of its relations. 
This is done by cutting across the trachea and adjoining" 
tissues as high in the neck as necessary or possible, and dis- 
secting them free from the cervical vertebrae and the first 
ribs. Then by drawing the trachea and surrounding tissues 
forcibly forward the aorta and overlying organs can be easily 
stripped from the vertebral column as low as the diaphragm. 
The left hand is now placed around the lower end of the 
pericardial sac, the aorta, and the esophagus just above the 
diaphragm, and the vessels are severed by cutting between 
the hand and the diaphragm. 

More space for the examination in situ of the vessels at 
the base of the neck can be obtained by freeing the clavicles 
from all attachments above and to the first ribs and drawing 
them forcibly outward ; this operation will be found especially 
useful in following up the subclavian vessels. . 

Removal of the I/ungs.— Pleural adhesions have al- 
ready been spoken of. If the base of the lung is adherent 
to the diaphragm, it is usually advisable to remove the latter 
with the lung by cutting through its insertion into the ribs. 
According to Orth, there is less danger of wounding the ab- 
dominal organs if scissors be used for the performance of 
the operation. After the lung is free it is drawn forward 
out of the pleural cavity, and the root of it is grasped from 
above downward between the separated fingers (first and 
second or second and third) of the left hand. The lung, 
thus resting in the palm of the left hand, is first drawn 
downward toward the pubes until the primary bronchus is 
divided by a nearly vertical incision above and behind the 
left hand. Then the lung is lifted vertically upward, and the 
rest of its attachments cut in the same direction from above 



POS T- MOR TEM EX A MINA TIONS. 



493 



downward by the knife held transverse and flat, so as to 
avoid injuring the esophagus and aorta. 

The procedure is the same for both lungs. Once in a 
great while the apex of a lung will be found so firmly adhe- 
rent by dense scar-tissue that it can be freed only by using 
the knife. 

The primary or main incision into a lung is a long, deep 




Fig. 160. — Method of incising the lung (Nauwerck). 



cut from the apex to the base and from the convex surface 
to the root, slitting the primary'bronchus, and thus not cut- 
ting it off from its branches to the upper and lower lobes 
(Fig. 1 60). To incise the left lung, place it with its inner or 
median surface and root downward on a board and with its 
base toward the operator. The left thumb steadies the lower 
lobe ; the first finger reaches between the two lobes almost 
to the primary bronchus ; and the rest of the fingers should 
hold the upper lobe. 



494 PATHOLOGICAL TECHNIQUE. 

The right lung is most easily incised by placing it in the 
same position, but with the apex toward the operator; in 
other words, always place the anterior edge of a lung beneath 
the palm of the hand. Some prefer to place each lung on 
its lower or diaphragmatic surface for incision. The right 
middle lobe is incised separately by a cut extending trans- 
versely in its greatest diameter. 

The bronchi and blood-vessels should be opened up for 
some distance with small probe-pointed scissors — as a rule 
from the surface of the section — cutting through the over- 
lying lung-tissue. In some cases, however, it is best to open 
up both the blood-vessels and the bronchi from the outside 
of the lung before incising it. The order to follow is vein 
first, then artery, and finally the bronchus. 

Secondary cuts into the lung are to be made parallel to 
the main incision. 

The bronchial lymph-nodes should be incised from the 
outside of the lung. 

Organs of the Neck. — The operation of the removal 
of the organs of the neck is greatly facilitated if it is pos- 
sible to continue the primary skin-incision up to the chin. 
In other cases dissect the skin from the larynx and muscles 
of the neck as far up as possible. In like manner free the 
muscles, esophagus, and trachea from their attachments later- 
ally and posteriorly. Then allow the head to drop well back 
over the end of the table, and pass a long, slender-bladed 
knife up between the skin and the larynx, just behind the 
symphysis of the lower jaw, until the point of the knife 
appears beneath the tip of the tongue. From this point the 
knife is carried with a sawing motion down first one ramus 
of the jaw and then the other, dividing laterally the glossal 
muscles as far back as the posterior pharynx. The knife is 
next carried up behind the esophagus, and the posterior wall 
of the pharynx divided as high as possible. Pass the left 
hand up inside of the neck and draw down the tongue. 
Then cut the attachments of the soft to the hard palate, 
carrying the knife well out so as not to injure the tonsils. 
Any remaining attachments are usually easily severed by 



POST-MORTEM EXAMINATIONS. 495 

pressing the tongue first to one side and then to the other, 
and cutting close to the roof of the pharynx. 

Each lobe of the thyroid gland is to be incised in its 
greatest diameter. 

Next cut through the middle of the uvula and examine 
all of the pharynx removed. Incise the tonsils vertically. 
The esophagus is to be slit in the median line posteriorly ; 
if it is normal, the larynx and trachea are then slit in the pos- 
terior median line also, thus splitting the esophagus in two. 

The Abdominal Cavity. — The order of removal of 
the abdominal organs varies with different operators, and 
under varying circumstances with the same operator. The 
gastro-intestinal tract, including the liver and pancreas, may 
be removed before or after the genito-urinary tract. The 
spleen as an organ by itself is often the first to be removed. 
The early removal of the liver is occasionally advantageous 
for the sake of the additional space obtained for the exami- 
nation of the other organs. It is well to practise the different 
methods of procedure, so that in a difficult case the best 
may be selected, because the examination of the abdominal 
cavity, especially in cases of extensive disease with numer- 
ous adhesions, is often one of the hardest tasks in post- 
mortem technique. As a rule, it is best to follow the usual 
order as long as possible, gradually removing the more or 
less normal or uninvolved organs. Occasionally it may be 
advisable to remove the organs en bloc, so as to be able to 
approach the problem from all sides. 

In all cases of acute peritonitis it is best before removing 
any organ to search for the source of the infection, paying 
particular attention to the vermiform appendix, to the gastro- 
intestinal tract, and, in females, to the pelvic organs. 

The order of removal of the abdominal organs adopted 
in this book for the majority of cases is that which seems 
the simplest and most natural — namely, to remove first the 
spleen as an organ essentially by itself; secondly, the gastro- 
intestinal tract, including the pancreas and liver, which forms 
the upper layer ; thirdly, the genito-urinary tract or middle 
layer, leaving the circulatory tract, the lowest layer, to be 
opened and inspected in situ. If, however, it proves neces- 



496 PATHOLOGICAL TECHNIQUE. 

sary to open a part of the gastro-intestinal tract in situ, it 
will be neater perhaps to remove the kidneys and spleen 
first. Occasionally at private autopsies it may be unneces- 
sary to examine the intestinal tract; under these circum- 
stances it is important to be able to get at the different 
organs without taking out the intestines. 

The Spleen. — As a rule, the spleen can easily be drawn 
forward from its bed behind the fundus of the stomach, be- 
neath the diaphragm, and lifted on to the lower edge of the 
ribs on the left side without cutting its vessels. The organ 
is then to be incised in its greatest diameter while thus firmly 
fixed between the left hand and the ribs ; or the vessels may 
be cut close to the hilus and the spleen incised after being 
placed on a board. 

In cases of adhesion to the diaphragm the spleen must be 
handled carefully while the fibrous attachments are torn or 
cut through, for the capsule is easily ruptured. Occasion- 
ally it is advisable to cut out with the spleen the portion of 
diaphragm attached to it. 

The important anatomical structures to be noted in the 
macroscopic examination are the capsule, trabecular, blood- 
vessels, lymph-nodules, and pulp. The weight of the spleen, 
according to Orth, varies from 150 to 250 grams. The 
average weight is put at 171 grams. The spleen measures 
12 X 7.5 X 3 cm. 

The Gastro-intestinal Tract. — The first step is to 
examine externally, more or less carefully according to the 
clinical symptoms, the whole tract from the stomach to the 
rectum, if it has not already been done at the primary in- 
spection of the peritoneal cavity. The main points to notice 
are distention or contraction of the intestines, injection of the 
blood-vessels, thickening of the wall, especially in the lower 
part of the ileum, adhesions, exudations, etc. Inspect the 
mesentery, its length, the amount of fat, and the size of the 
lymph-nodes ; incise the latter to determine color and con- 
sistency. Examine the mesenteric vessels if any evidence 
of infarction of the intestine is noticed. The portal vein and 
its branches should be opened up in situ, in all cases of ab- 



POST-MORTEM EXAMINATIONS. 497 

scess of the liver or of secondary deposits in it of malignant 
growths, before the gastro-intestinal tract is removed. As a 
rule, it is not necessary to open any part of the gastro-intes- 
tinal tract in situ. The operation can be performed much 
more neatly at the sink. The duodenum is often opened for 
the sake of investigating the flow of bile from the gall-duct, 
but except in cases of jaundice the operation must be looked 
upon largely as a physiological experiment. 

Free the omentum from the transverse colon by putting it 
on the stretch and dividing it with the knife close to the 
colon. Then begin the removal of the large intestine by 
drawing the sigmoid flexure forcibly forward and cutting the 
mesocolon close to the gut, first down to the rectum, then 
upward to the transverse colon. Free the latter by dividing 
the two folds of the lesser omentum, if not already cut 
through, which unite it to the stomach. The ascending 
colon is to be freed in the same manner as the descending 
portion. Care should be taken not to injure the appendix. 
If the lower part of the sigmoid flexure be now stripped up- 
ward a short distance with the fingers, so as to force the in- 
testinal contents out of the way, the gut can be divided just 
above the rectum without fear of the feces escaping. 

Place the freed intestine in a pan or pail, and as the small 
intestine is divided from its mesentery deposit it in the same 
receptacle. To remove the small intestine, begin at the ce- 
cum, and, while lifting the ileum with the left hand strongly 
enough to keep the mesentery constantly tense, cut the latter 
close to the intestine by playing the knife easily backward 
and forward across it with a fiddle-bow movement. Con- 
tinue the operation until the duodenum is reached. The 
mesentery can now be dissected from the duodenum and re- 
moved, or the mesentery, duodenum, pancreas, and stomach 
can be removed in continuity with the intestine by carefully 
dissecting them off the underlying structures. The opera- 
tion is perhaps more easily accomplished by freeing the 
organs from below upward. First cut down through the 
diaphragm and free it around the esophagus. Then separate 
the stomach from the liver by means of the thumb and fingers 



498 PATHOLOGICAL TECHNIQUE. 

of the left hand in such a way as to put on the stretch the 
vessels of the hepato-duodenal ligament. These vessels 
(hepatic artery, common gall-duct, and portal vein) are then 
carefully divided in the order named. As each vessel is cut 
the character of its contents should be observed to see if 
anything abnormal is present. 

The mesentery, if still present, the duodenum, the pan- 
creas, and the stomach, are now to be dissected carefully 
away from the underlying vessels from below upward until 
the esophagus is reached. This may be constricted by the 
fingers at any point desired, and cut across without danger 
of the gastric contents escaping and without the necessity 
of tying. In certain cases of hemorrhage from the stomach 
associated with cirrhosis of the liver it is important to re- 
move the esophagus in continuity with the stomach, because 
in these cases the hemorrhage usually takes place from 
dilated esophageal veins. 

The stomach and intestines are now to be opened at the 
sink by means of the enterotome, the colon along one of its 
longitudinal muscular bands, the small intestine along its 
mesenteric attachment, because the most important lesions 
usually occur opposite this line in the lymph-nodules and 
Peyer's patches. The stomach is opened by many along the 
greater curvature ; others, however, prefer to cut along a 
line 3 cm. from the lesser curvature, on the ground that 
better museum preparations are thus obtained. In case 
any tumor or focal lesion is perceived from the outside, it 
is advisable to cut the stomach, if possible, in such a way as 
to leave the pathological part uninjured. 

Whenever jaundice is present the duodenum must be 
opened in situ in order to examine the bile apparatus in con- 
tinuity, so as to determine whether the coloring is due to 
obstruction of the hepatic or common gall-ducts, or is of so- 
called hematogenous origin. 

To open the duodenum make a transverse fold in the ante- 
rior wall and incise with the scissors. Continue the longi- 
tudinal slit thus made up as far as the pylorus and down to 
where the duodenum passes beneath the mesentery. Notice 



POST-MORTEM EXAMINATIONS. 499 

the contents of the duodenum and their color both above 
and below the opening of the gall-duct. The ductus cho- 
ledochus usually opens in common with the ductus pan- 
creaticus on the posterior wall of the duodenum a little 
below the middle of the head of the pancreas, at a point 
marked by a small papilla which can easily be recognized 
by putting the mucous membrane on the stretch transversely. 
Press first on the common duct gently and in the direction 
of the papilla, watching the opening to see if any obstructing 
material is forced out. Pressure is then to be made on the 
gall-bladder to see if its contents also will flow. If neces- 
sary, the common duct and its branches are to be opened in 
situ. In certain cases the ductus pancreaticus is likewise to 
be opened up. 

Several cross-sections of the pancreas are usually better 
than one in the greatest diameter, because the duct is left in 
a better condition for slitting up if necessary. The weight 
of the pancreas varies from 90 to 120 grams (Orth). It 
measures 23 X 4.5 X 2.8 cm. 

The Liver. — The liver is usually the last organ of the 
gastro-intestinal tract to be removed. This is ordinarily 
done by lifting up the right lobe and freeing it from all 
attachments as far as the vertebral column : the right lobe is 
then lifted and placed on the edge of the ribs on the right 
side, while the left lobe is elevated and freed. If the dia- 
phragm is firmly adherent, remove it with the liver. The 
incision to display the liver is a long deep cut passing through 
the right and left lobes in the greatest diameter of the organ. 

In a good many cases it is very convenient to remove the 
liver at the beginning of the special examination of the ab- 
dominal cavity, because more room can be obtained for the 
investigation of the other organs. This latter fault can to 
some extent be obviated by cutting the diaphragm on the 
right side and allowing the liver to slide forward somewhat 
into the right thoracic cavity. 

There can be no objection to the removal of the liver 
when jaundice is not present or when the liver is not con- 
nected by continuity with the lesion of some, other organ 



5PO PATHOLOGICAL TECHNIQUE. 

(pylephlebitis, malignant growth extending through portal 
vein or along gall-ducts, etc.). 
! The operation is performed as follows : Pass the left hand 
in between the diaphragm and the right lobe and push the 
liver forward out of the right hypochondrium. Incise it 
deeply in ; its greatest diameter through the left and right 
lobes. Next free the gall-bladder from its bed by means of 
the fingers, and cut it off near the ductus hepaticus after 
compressing its lower end. It can then be opened length- 
wise and washed without danger of discoloring the liver or 
other organs. The liver is now to be grasped by placing 
the thumb on the under surface of the liver and the fingers 
in the incision. Elevate the organ, and, while carefully 
watching, cut through the hepato-duodenal ligament, which 
includes the blood-vessels and the ductus hepaticus. The 
ligamentum' hepato-gastrium, the inferior vena cava, the 
suspensory ligament, the ligamentum coronariufn, and the 
tissue between the inferior surface of the liver and the upper 
end of the kidney follow next : the adrenal is to be left on 
the kidney, and the diaphragm ought not to be injured. 

Even in the ordinary way of removing the liver the organ 
will be found much easier to handle if the usual incision is 
made in situ, so as to furnish a hold for the left hand. 

Other cuts into the liver are best made parallel to the 
primary one. 

Orth gives the weight of the liver for adults as varying 
from iooo to 2000 grams. The average weight is usually 
put at 1500 to 1800 grams. 

The liver measurements are as follows : 

Length from right to left . . . , 2 5~3 2 cm - 

Width of right lobe 18-20 

Width of left lobe 8-10 

Vertical diameter of right lobe 20-22 

Vertical diameter of left lobe 15-16 

Greatest thickness 6- 9.5 

The Kidneys and Adrenals. — If the adrenals are to be 
removed with the kidneys, it is necessary to cut first to the 
inside, and secondly above the adrenal, and then to make 



POST-MORTEM EXAMINATIONS. 50I 

from the outer end of the second cut a curved incision alonig 
the outer convex border of the kidney through the perito- 
neum and the perinephritic fat-tissue. The left hand is to 
be inserted into the cut, the mass of tissue drawn forcibly- 
forward, and the vessels divided as close to the aorta as pos- 
sible, so that the renal vessels may be slit up and examined 
in connection with the renal lesions. The adrenal should be 
incised crosswise. The kidney is to be held firmly in the 
left hand between the thumb and fingers while a longitudinal 
incision is made from the convex border to the hilus. As a 
rule, it is better to shell it out of its investing fat-tissue before 
incising it. 

It will often be found convenient to make simply the curved 
incision above given, to shell the kidney out of its fat-cap- 
sule, and then to divide its vessels, leaving the adrenal behind 
to be incised in situ or removed separately. As a rule the left 
kidney is removed first. 

In all cases in which the bladder is involved in patho- 
logical changes in common with the kidneys the whole 
urinary tract should be removed intact, so that the lesions 
may be examined in continuity. For this reason it is a good 
plan to open up the pelvis of the kidney and the ureter from 
the primary incision, in order to see if any lesion is present 
before dividing the ureter. 

If it is desired to remove the kidneys before the intestines, 
the latter must to some extent be freed from their normal 
attachments. 

The splenic flexure of the colon is first to be drawn for- 
cibly forward and its attachments divided where they hide the 
left kidney. If the ureter is to be taken out also, it is best 
to free the whole of the descending colon from its mesocolon. 
Then the colon and the coils of small intestine are drawn 
over to the right side of the body, so as to leave the left kid- 
ney and adrenal exposed. They are then removed in exactly 
the same manner as already described. 

To remove the right kidney the hepatic flexure must be 
freed from over it. If the ureter is to be taken out, the de- 
scending colon and the cecum are dissected from over it. 



502 PATHOLOGICAL TECHNIQUE. 

The right adrenal is firmly attached to the under surface of 
the liver, and must be carefully dissected from it by turning 
the latter upward. 

If the urinary tract is to be removed in continuity, each 
ureter is dissected down to the brim of the pelvis, and then 
left with its kidney attached until the pelvic organs have 
been taken out. 

After the kidney has been incised the capsule is to be 
stripped off, at least in part, so that the appearance of the 
surface of the kidney and the presence or absence of adhe- 
sions between the capsule and the renal tissue can be deter- 
mined. 

The points to be noted in the macroscopic examination of 
the kidney are size, consistency, and, on section, color, rela- 
tive proportion of cortex to pyramids, and thickness of each ; 
finally, the normal markings of the kidney, including blood- 
vessels, glomeruli, convoluted and straight tubules of cortex, 
collecting tubules of pyramids. 

The average weight of the kidney is 150 grams. The left 
kidney is always 5 to 7 grams heavier than the right (Orth). 
A kidney measures 1 1-12X5-6X3-4.5 cm. The cortex 
measures in thickness 4-6 mm. The relation of the cortex 
to the medulla is 1 to 3. 

The Pelvic Organs. — The pelvic organs are most easily 
and neatly removed by stripping the peritoneum from the 
pelvic wall with the fingers. Begin over the bladder and 
extend down the sides of the pelvis until the fingers meet 
beneath the rectum. Brace the backs of the hands laterally 
on the brim of the pelvis and lift the fingers forcibly upward ; 
this movement will free the pelvic organs cleanly from the 
sacrum, and leave them attached only anteriorly at the rectal 
and genital openings, and posteriorly by the peritoneum and 
the vessels at the brim of the pelvis. 

Anteriorly, the attachments may now be divided with the 
knife at whatever point seems advisable, ordinarily close to 
the pubes just anterior to the prostate (or through the ure- 
thra and vagina in females) and through the lower end of 
the rectum. Posteriorly, cut through the tissues at the brim 



POST-MORTEM EXAMINATIONS. 503 

of the pelvis, taking care not to cut the ureters if the kid- 
neys are still attached to them. The rectum is to be opened 
with the enterotome along the posterior wall, and the inner 
surface thoroughly washed off so as to avoid soiling the 
other organs. 

To open the bladder in males, especially if the penis has 
been removed in continuity with it, incise with the scissors a 
transverse fold in the anterior wall of the fundus, and carry 
the incision through the urethra and along the dorsum of the 
penis. To accomplish the latter act perfectly the penis must 
be firmly stretched by having an assistant pull at the frenum 
while the bladder is held fixed by the operator. 

In females it is usual to enter the scissors into the bladder 
through the urethra and to cut through the middle of the 
anterior wall of the fundus. 

In males the rectum should be dissected from the bladder, 
so as to lay bare the vesicular seminales and the prostate, 
which are examined by means of several transverse incisions. 

In females, if the bladder is normal, the vagina is incised 
in the anterior wall through the middle of the bladder. Or 
the vagina may be incised laterally until the cervix is reached, 
and then the cut be carried up to the median line. 

The uterus is incised in its anterior wall from the cervix 
to the fundus. From the upper part of this incision second- 
ary incisions are carried out on each side to the orifices of the 
Fallopian tubes. 

The ovaries are incised in their greatest diameter, from the 
convex border to the hilus. Weight of ovaries, 7 grams. 

The testicles can readily be examined without external 
injury to the scrotum by cutting underneath the skin over 
the pubes down to the scrotum on either side of the penis, 
and shoving the testicles up through the incision. Cut care- 
fully through the overlying tissues until the cavity of the 
tunica vaginalis is opened. Remove the testicle by severing 
the cord. The incision to display a testicle should be in the 
long diameter, beginning on the side opposite the epididymis 
and extending through into it. Weight of testicles, 1 5-24.5 
grams. In cases of tuberculosis of the testis and epididymis 



504 ... PATHOLOGICAL TECHNIQUE. 

it is advisable not to cut through the cord, but to remove 
the testicles and cords with the bladder, so that the whole 
genital tract may be examined in continuity and the asso- 
ciated lesions in the vesiculae seminales demonstrated, if 
present. 

The penis, or at least the larger portion of it, can be re- 
moved in connection with the bladder by continuing the pri- 
mary body-incision out to about the middle of the dorsum 
of the penis, which is then to be freed from the investing 
skin and divided just posterior to the corona. It is next 
dissected back to the pubic arch, and freed from it partly by 
cutting from without, partly from within, the pelvis, until the 
penis can be passed underneath the arch into the pelvis. 
Other methods are to cut through the symphysis, which can 
then readily be sprung apart by swinging one of the legs 
out in a horizontal plane, or even to saw out a small sec- 
tion of bone including the symphysis, so as to have more 
room for freeing the attachment of the penis and for re- 
moving it. 

The structures now remaining in the abdominal and 
thoracic cavities which require examination are the large 
blood-vessels, the thoracic duct, the celiac ganglion, and the 
retroperitoneal lymph-nodes. The inferior vena cava and 
its branches are first examined (especially in all cases of 
pulmonary embolism) by slitting them with scissors along 
the anterior wall. If it is necessary to follow the iliac ves- 
sels into the thigh, it will be found easier in sewing up if the 
primary abdominal incision is continued off to the side in 
question, thus giving a single though curved incision. 

It is sometimes advisable to open up the inferior vena cava 
and its branches before removing the pelvic organs, so that 
thrombi extending into the pelvic vessels may be examined 
before they are disturbed. 

The semilunar ganglia lie on the aorta, around the celiac 
axis, above the pancreas. 

The thoracic duct lies behind and to the right of the aorta. 
In the thorax it is most easily found by dissecting on the 
right side between the aorta and the azygos vein. The re- 



POST-MORTEM EXAMINATIONS. 505 

ceptaculum chyli lies to the right and behind the aorta upon 
the second or third lumbar vertebra. Examination of the 
thoracic duct is of especial importance in cases of tubercu- 
losis of the intestine and mesenteric lymph-nodes with 
secondary miliary tuberculosis. 

The aorta is to be opened in situ along the anterior wall 
throughout its whole extent, and the iliacs as far as the 
femoral ring. 

Besides the brain, the spinal cord, and the thoracic and 
abdominal organs, it is often necessary to examine or remove 
for study other portions of the body that are affected by dis- 
ease. A little ingenuity will enable one in appropriate cases 
to get at almost any part desired. 

A* view of the marrow in a long bone is most easily ob- 
tained in the femur by extending the body-incision down 
over one of the thighs, dissecting the muscles way, and then 
chiselling off a portion of the upper part of the shaft. 

In tuberculosis of the spine it is quite easy to remove any 
part, or even the whole, of the vertebral column, including 
the pelvis and portions of the femurs, without other incisions 
than the one from the neck to the pubes, with extension 
down the thighs in case parts of the femurs are to be taken 
out. Divide the ribs a few centimeters from the vertebral 
column on each side of the portion that is to be removed, 
cut through intervertebral disks both above and below it, 
and then carefully dissect it free, taking great care not to 
button-hole the skin. 

Removal of the Brain. — The incision into the scalp 
should begin from one to two centimeters behind the right 
ear, near its lower border, at the edge of the hair, and ex- 
tend over the vertex of the skull to a corresponding point 
behind the left ear. The cut is most easily made by thrust- 
ing a small narrow-bladed scalpel, with its back toward the 
calvarium and its point toward the vertex, through the skin 
behind the ear and shoving it along in the desired direction. 
By making the incision in this manner the hair is not cut, 
but simply parted. The anterior flap should be stripped 
from the calvarium and the temporal muscles by putting it on 



506 PATHOLOGICAL TECHNIQUE. 

the stretch and dividing the loose connective tissue hold- 
ing it by sweeping strokes of the scalpel nearly as far for- 
ward as the orbits. After a part of the flap has been freed 
it is often possible to strip the rest without using the scalpel. 
For the posterior flap, which should be removed back as far 
as the occipital protuberance, the scalpel nearly always has 
to be used. 

If the hair is long, the anterior portion can be rolled into 
the anterior flap over the face and thus protected. The 
posterior portion is gathered at the nape of the neck, and 
then a towel is wrapped tightly around the head and neck, 
extending from the line where the flaps are reflected down 
to the shoulders, and is pinned over the lower part of the 
forehead. In this manner the hair is perfectly protected from 
being soiled and ample room is left for work. 

Of the two methods of opening the skull, the circular and 
the wedge-shaped, the former makes the better museum 
preparation, but the latter is in greater use in this country, 
and has the advantage of rendering the calvarium less likely 
to slip out of place after the head has been sewed up. 

The wedge-shaped incision consists of three cuts, which 
should be outlined on the periosteum of the skull with a 
scalpel. The first cut begins just above and behind the left 
ear, and is carried over the forehead just back of the edge 
of the hair or over the frontal eminences to a corresponding 
point above and behind the right ear. The two other cuts 
begin at each end of the first incision, forming there an 
obtuse angle, and are carried back to meet in the median 
line behind at an angle of about i6o° a little in front of the 
occipital protuberance. The temporal muscle on each side 
is now to be scraped back from the line of incision out of 
the way of the saw, but is not to be cut off. The holder, 
if one is used, is attached with a foot in each obtuse angle 
in the temporal region. If a holder is not employed, the 
head is best steadied by hands on the calvarium and face. 
Use towels or cloth to prevent slipping. 

Start the incision with the saw over the forehead and 
extend it back along the line marked out. It is best not to 



POST-MORTEM EXAMINATIONS. 



5°7 



carry the incision clear through the inner table of the bone, 
for two reasons : first, on account of the danger of injuring 
the brain-substance ; secondly, because if the inner table or 
a part of it is cracked through with a chisel and hammer, it 
can be done without injuring the underlying tissue, and the 
irregular overlapping fragments of bone thereby formed 
serve afterward for holding the calvarium firmly and steadily 
in place. 

After sawing along the lines marked out, insert a chisel in 
the frontal region, and with a quick, sharp blow crack 
through the rest of the inner table. In like manner insert 
the chisel in the middle of the other incisions and free the 
calvarium posteriorly. To remove the calvarium insert the 
chisel end of the hammer in the incision in the frontal 
region, and press down with the left hand while swinging 
the handle around in a horizontal plane. 

By means of the powerful purchase obtained the calvarium 
is easily started. Then catch the hook of the hammer over 
the calvarium and strip it off. If the dura is adherent to the 
calvarium, it may be freed by using the point of the closed 
enterotome to pry it off. 

In young children, and sometimes in old people, it is 
necessary to remove the dura with the calvarium. To do this, 
cut through the dura with the point of a scalpel along the 
lines of incision in the skull ; then cut the falx cerebri in 
the median line, both anteriorly and posteriorly. 

An infant's skull is best opened by cutting with a pair of 
scissors through the dura along the sutures (in the longi- 
tudinal suture on each side of the falx) well down to the 
floor of the skull. This gives five bone-flaps which may be 
turned out like the petals of a flower, leaving the brain unin- 
jured. It is often necessary to cut half of the base of each 
flap in a horizontal line to aid its being turned out. The 
falx cerebri must of course be divided anteriorly and drawn 
back before the brain is removed. In sewing up, the bone- 
flaps are turned in over a bag of sand or sawdust filling the 
cranial cavity, and are kept perfectly in place by the skin. 

In a case of fracture of the skull no cracking with hammer 



508 PATHOLOGICAL TECHNIQUE. 

and chisel is allowable ; the calvarium must be freed entirely 
by sawing. The calvarium should be examined at the time 
of removal. 

The next step is to inspect the dura. Under normal con- 
ditions it is not tense in the frontal region, but can be picked 
up with the forceps or fingers. If the dura is not thickened, 
the convolutions normally should be visible through it. The 
longitudinal sinus is opened with knife or scissors and its 
contents examined. Pacchionian granulations are not infre- 
quently found projecting into it. 

To remove the dura, cut through it with scissors or knife 
along the same lines in which the calvarium was sawn. Turn 
back each half of the dura and examine the surface of the 
convolutions and the inner surface of the dura. The con- 
volutions should be distinct and rounded, not flattened, with 
obliteration of the gyri, as occurs when there is internal 
pressure. 

The Pacchionian granulations are situated along the longi- 
tudinal fissure and may grow through the dura and form 
depressions in the calvarium. There may be apparent adhe- 
sions between the dura and pia due to veins passing from 
one to the other. The dura is still further freed by seizing 
the two halves anteriorly and lifting them up until the falx 
is tense at its insertion into the crista galli. Pass a knife in 
parallel to the falx, on the left side, with the edge forward, as 
far as the cribriform plate ; turn it to the right and cut until 
the falx yields. Withdraw the knife in the same manner in 
which it was inserted. Next draw the dura back. It is usu- 
ally more or less attached along the longitudinal fissure by 
Pacchionian granulations and by blood-vessels. These may 
be cut or torn through. Do not cut the dura posteriorly, 
but let it hang down. 

To remove the brain, insert the two fore fingers, or the 
first and second fingers of the left hand, anteriorly between 
the dura and the frontal lobes, one on each side of the falx 
cerebri, and draw the brain gently back until the optic nerves 
are visible. Ordinarily, the olfactory nerves come away from 
the cribriform plate without trouble, but sometimes have to 



POST-MORTEM EXAMINATIONS. 



5°9 



be freed with the point of the knife. With a long, slender- 
bladed knife divide the optic nerves as far forward as possible 
while holding the brain back with the left hand. Continue 
to draw the brain carefully back and divide the cranial nerves 



Hypoph 



N. trigem 

N. facial. 

N. acust. 




Carotis int. 



S. cavernos. 



Ifl- S. petr. inf. 
S.petr. sup. 
Cut edge 
of tento- 
rium. 



N. glossophar. 
N. vagus 
N. recurr. 



N. hypogl. 
Tentorium 



S. transvers. 
A. vertebral, 
occipit. 



Fig. 161. — Base of skull (Nauwerck). 

and the carotids. Then draw forward first the left, then the 
right temporal lobe, and cut the tentorium close to its attach- 
ment to the petrous portion of the temporal bone with a 
sawing motion, using the tip of the knife. Insert the knife 
at the side close to the squamous bone, and cut from there 



5IO PATHOLOGICAL TECHNIQUE. 

in toward the foramen magnum. Then cut the nerves given 
off from the medulla oblongata while supporting the con- 
vexity of the brain in the left hand. 

Lastly, carry the knife as far as possible into the spinal 
canal, and divide the cervical cord by an oblique incision 
from each side, severing the vertebral arteries with the same 
stroke. Better than a knife is the myelotome, because it 
gives a cross-section of the cord and allows more of it to be 
removed. 

The brain is now to be removed by passing the first and 
second fingers of the right hand in on either side of the cord, 
and everting the brain while still supporting it posteriorly 
with the left hand. 

Before proceeding to open the brain it is best to examine 
the base of the skull, particularly the dura, of which the 
sinuses should be incised, and the hypophysis cerebri. 

If there is. a suspicion of a fracture at the base, strip off the 
dura, so as to give a better opportunity for examination of 
the bone. 

The brain should be weighed before it is dissected. The 
average weight in an adult male is 1358 grams; in an adult 
woman, 1235 grams. 

External Examination of the Brain. — Place the 
brain with the base uppermost and with the cerebellum to- 
ward the operator. Examine first the pia and the cranial 
nerves, then the arteries, especially the middle cerebral and 
its branches on each side in the fissure of Sylvius, for it is 
here that emboli most frequently lodge. The pia bridging 
the fissure of Sylvius can sometimes be torn through, but 
usually has to be cut. 

It is important, particularly in cases of obscure cerebral 
symptoms, to feel gently with the finger-tips all over the sur- 
face of the brain for any areas of increased density, because 
patches of sclerosis may in that way be found which might 
otherwise be overlooked. 

By stripping off the pia — a procedure not often advisable 
— -adhesions over pathological areas can sometimes be found 
pointing to the lesions beneath, but the pia should not be 



POST-MORTEM EXAMINATIONS. 511 

stripped from those portions which are to be examined mi- 
croscopically. To remove the pia an incision is made on the 
median surface of each hemisphere just above the corpus 
callosum from one extremity to the other, and the pia 
stripped back first from the median and then from the con- 
vex surface. The stripping is done by means of the fingers, 
with occasional aid from the forceps. 

Section of the Brain. — There are several methods of 
cutting up the brain, no one of which is particularly suitable 
to all occasions. That method must be chosen which is most 
fitted to the individual case and to the use to which the 
tissue is to be put. 

The ideal method from a neuro-pathological standpoint 
would undoubtedly be to harden the brain entire, and then 
to make serial frontal sections thin enough for microscopical 
purposes through the whole organ. The nearest approach 
to this ideal method is to harden the brain entire in formal- 
dehyde, a process occupying ten days to two weeks (see 
page 121), to make thin serial sections, to mordant the sec- 
tions, divided if necessary into smaller pieces, in a chrome 
salt (preferably by Weigert's quick method), and then to 
carry through a number of series from the important parts 
for microscopical examination. By this means the relations 
of the various cerebral structures and of the pathological 
lesions can be perfectly preserved and studied. This method 
can be particularly recommended for tracing degenerations 
in the motor tract. 

If there is a noticeable focal lesion, such as a tumor or 
hemorrhage, it should be so incised, generally frontally or 
horizontally, as best to show its relations to the important 
cerebral tracts and ganglia. In these cases also the best re- 
sults are obtained by hardening the brain entire in formal- 
dehyde, and later making serial sections for macroscopic 
study or for carrying through for histological purposes. In 
many cases, however, it is necessary or advisable to examine 
the lesions in the fresh state. For instance, if it be desired 
to study the neuroglia-fibers, it is positively necessary to cut 
out thin slices of fresh tissue and to fix them immediately in 



5i2 



PA THOL O GICAL TE CHNIQ UE. 



the proper solution. Often, too, the lesion cannot be or is 
not found except on fresh examination, or the clinician whose 
case it is desires to see at once the cause of certain symp- 
toms. Under such circumstances the more ideal method 
must be sacrificed, and as much made out of the case as is 
possible in the condition in which it is left after the exami- 
nation. 

For the routine examination of the brain, to demonstrate 

G.forn. G. front. I. 
S. call marg. J G. front. II. 

G. front. III. 



G. centr. a. 
S. Rol. 
G. centr. 4>. 




marg. 
ang. 
S. interpar. . 
par. sup. 

par. occ. 
C. call. 
Fig. 162. — First cut in the brain (Nauwerck). 

its topography and to bring to light suspected or unsus- 
pected lesions, probably no method is more generally used 
than Virchow's. The objection most often made against it 
is that the cerebral cortex is too much cut up. In case, 
however, it is desired to preserve the cortex or parts of it for 
microscopic purposes, the longitudinal incisions after the first 
may be omitted, and the cortical portion, after being sepa- 
rated from the stem, may be cut in any way that seems ad- 
visable. In like manner, the brain-stem or any other part 



POST-MORTEM EXAMINATIONS. 513 

may be left uncut, and hardened entire in formaldehyde for 
histological purposes. 

Virchow's Method. — The brain is to be placed on its base 
in the same position as one's own. Press the hemispheres 
apart a little so as to expose the corpus callosum. Hold the 
left half of. the cerebrum in the left hand with the fingers on the 
lateral aspect and the thumb in the longitudinal fissure. Then 
make an almost vertical incision with a long, slender knife 
through the roof of the left ventricle in its middle third, 2 to 
3 mm. from the median raphe of the corpus callosum. The 
roof of the ventricle is to be slightly raised vertically by the 
thumb, so that the incision, which must not be too deep, may 
not injure the basal ganglia. The incision is to be continued 
into the anterior and posterior cornua. Then make a long 
incision from one end of the above cut to the other, passing 
just outside of the basal ganglia at an angle of about 45 °. 
Repeat the process on the right side, turning the brain half 
around. Next seize what remains of the corpus callosum 
and fornix in the middle, lift them, and cut through from be- 
low up, passing the knife through the foramen of Munroe. 
The parts are then turned back, exposing the velum inter- 
positum and the choroid plexuses. By drawing back the 
velum interpositum the third ventricle is uncovered. 

The corpora quadrigemina are exposed by cutting trans- 
versely the right posterior pillar of the fornix and adjoining 
brain-substance and carrying them over to the left. Each 
ventricle as it is opened is to be carefully inspected and any 
abnormal condition of its ependyma noted. The cortex is 
further divided on one side, and then on the other, by hold- 
ing it in the left hand and making vertical straight sections 
from the upper angle of the previous cut into the convex 
cortex, allowing the sections to fall apart, so as to avoid 
touching and soiling the surface with knife or fingers. Each 
portion thus cut represents a prism. The incisions should 
go well into the cortex, but not so far as to separate the 
different pieces. The basal ganglia are examined by means 
of a number of frontal sections. For this purpose the left 
hand is placed palm upward underneath the brain, so that as 



5*4 



PA TH0L0G1CAL TECHNIQUE. 



each section is made over the tips of the fingers by one long 
stroke of the knife it falls forward, exposing a clean surface 
of which the two halves can be compared. An incision is 
next carried through the middle of the pineal gland, the 
corpora quadrigemina, and the vermiform process of the 
cerebellum, opening the aqueduct of Sylvius and the fourth 
ventricle. 

Each half of the cerebellum is divided by a median hori- 



Corp. call. 
Corp. striat. 



Corp. call. 




Cr.forn. desc 



Cr. forn. desc. 
Fig. 163. — Section of the brain (Nauwerck). 



zontal section into halves, and these portions are still further 
subdivided by a series of cuts radiating from the peduncles. 

In order to make sections of the pons and medulla the 
brain is folded together and turned over. Several cross- 
sections are then made with the left hand placed beneath as 
in sectioning the basal ganglia. 

Before making the sections it is well to remove the basilar 
and vertebral arteries, especially if they are calcified. 

In Pitre's method of dissecting the brain the lateral ven- 
tricles are opened as in Virchow's method. Then the pedun- 
culi cerebri are cut squarely across, so as to remove the pons 



POST-MORTEM EXAMINATIONS. 515 

and cerebellum, and a longitudinal incision is carried down 
through the third ventricle, halving the cerebrum. Through 
each half of the cerebrum a series of six sections is then 
made parallel to the fissure of Rolando. The names of the 
sections and the important parts which they show are as 
follows : 

1. The pre-frontal section through the frontal lobe, 5 cm. 
anterior to the fissure of Rolando, shows the gray and white 
substance of the frontal convolutions. 

2. The pediculo-frontal section through the posterior por- 
tions of the three frontal convolutions shows the anterior 
extremity of the island of Reil, the lenticular and caudate 
nuclei, and the internal capsule. 

3. The fro7ital section through the ascending frontal con- 
volution, parallel to the fissure of Rolando, shows the optic 
thalamus, the lenticular and caudate nuclei, the claustrum, 
the external and internal capsules, the anterior portion of the 
descending horn of the lateral ventricle, and the island of 
Reil. 

4. The parietal section through the ascending parietal con- 
volution shows portions of the same structures as the pre- 
ceding, and a transverse view of the hippocampus. 

5. The pediculo-parietal section through the parietal lobe, 
3 cm. posterior to the fissure of Rolando, shows the tail of 
the caudate nucleus in two places and the posterior portion 
of the optic thalamus. 

6. The occipital section through the occipital lobe, 1 cm. 
in front of the parieto-occipital sulcus, shows simply the 
white and gray matter of the occipital lobe. The cere- 
bellum, pons, and medulla are incised in the manner already 
described. 

Removal of the Spinal Cord. — The body is to be 
placed face downward, with the head over the end of the 
table and a block under the chest. The incision is made 
over the spinous processes from the occiput to the sacrum. 
Dissect the skin and muscles back on each side, so as to 
leave the vertebral laminae as bare as possible. The laminae 
may be cut through by means of several instruments, of 



51 6 PATHOLOGICAL TECHNIQUE. 

which the double-bladed saw (Luer's rhachiotome) is perhaps 
tbe safest, at least for beginners. The single-bladed saw 
with rounded end is also very useful and can be thoroughly 
recommended. The operation can be done most quickly by 
biting off the spinous processes with the heavy bone-forceps 
and cutting through the laminae with chisel and hammer, 
but there is greater danger of injuring the cord. 

' The numerous artifacts in the cord, reported as neuromata 
and heteroplasia even within very recent times by competent 
pathologists, would seem to indicate that the need of careful 
and delicate technique in the removal of the spinal cord is 
not yet fully appreciated. 

The laminae should be sawn nearly or entirely through in 
a line with the roots of the transverse processes from the 
third or fourth lumbar vertebra to the cervical region. The 
arches of the cervical vertebrae are best divided with a heavy 
bone-cutter, because they cannot be easily sawn, and there 
is sufficient room here for the points of the bone-cutter with- 
out danger of their pressing on the cord. 

= It is important to strike the outside limits of the spinal 
canal, so as to get as much room as possible for the removal 
of the cord. Test if the sawing be deep enough by the 
mobility of the spinous processes. If necessary, they can 
be freed by means of the hatchet-chisel and a hammer in 
the same way that the calvaria is loosened. 

As the cord reaches only to the second lumbar vertebra, 
cut through between the third and fourth, free with the 
heavy bone-cutter the lower end of the row of the spinous 
processes, which are held together by their ligaments, and 
strip them up to the neck ; then cut through the cervical 
arches with the bone-cutter, taking care that the point 
within the canal does not come in contact with the cord. 
! The nerve-roots are to be divided with a sharp scalpel by 
means of a long cut on each side of the cord. Then cut 
across the dura and the nerve-roots at the lower end of the 
exposed canal, and, while holding the dura with forceps, 
carefully free the cord from below up with scissors or scalpel, 
taking care , all, the time not to pull or bend the cord, be- 



POST-MORTEM EXAMINATIONS. '*$J 

cause in either way artifacts may be produced. Cut the Cord 
squarely across as high in the cervical canal as possible, s6 
that the remaining portion may be easily removed with 1 tlie 
brain. 

Lay the the cord after removal on a flat surface and incise 
the dura longitudinally, first posteriorly and then in front. 
A series of cross-sections, usually I to 2 cm. apart, is made 
through the cord while supported on the fingers during the 




Fig. 164. — Base of skull, showing lines of incision for removing internal eye,, etc 

(Nauwerck). 

cutting, so that the cut surfaces shall fall apart. The dif- 
ferent segments should ordinarily be left attached to the 
dura, so that their position in the cord can easily be deter- 
mined. 

A diagnosis from the fresh, macroscopic appearances of 
the cord is often very difficult to make, according to the best 
authorities. : ' 

The Eye. — The contents of the orbit, including the poste- 
rior part of the eye, can be readily examined by chiselling 



518 PATHOLOGICAL TECHNIQUE. 

off the roof of the orbit. The posterior half of the eye can 
be removed by cutting around the eyeball with sharp scissors 
without changing the hold of the forceps on the sclera. If 
done quickly, the retina remains quite well spread out. The 
anterior half of the eyeball is to be propped in place by a 
plug of cotton dipped in ink or in a solution of perman- 
ganate of potassium. 

The Ear. — The middle ear can be exposed by chipping 
off with a chisel its roof, which lies in the middle of the 
petrous portion of the temporal bone. The roof can also 
be very easily bitten off with the heavy bone-cutters. If, how- 
ever, it be desired to examine the ear more carefully by 
means of a section through the external meatus and the 
aniddle ear, it will be necessary to remove the whole of the 
petrous bone. For this purpose the incision behind the ear 
must be carried back along the anterior edge of the trapezius 
muscle halfway down the neck. Then the skin-flaps, in- 
cluding the external ear and the underlying tissues, must be 
dissected back for some distance on each side of the incis- 
ion. Two converging incisions are then to be sawn, the 
anterior passing through the root of the zygomatic arch, 
the posterior just back of the sigmoid sinus, so as to come 
together at the apex of the pyramid of the petrous bone, or, 
better still, to meet in the foramen magnum. An ordinary 
chisel and a hammer or mallet will be found very convenient 
for freeing the petrous bone after the incisions have been 
sawn. 

In the examination of the petrous bone after it has been 
removed the first step is to chisel off the tegmen tympani so 
as to get a view of the middle ear. Next remove the lower 
wall of the external meatus, so as to expose the outer sur- 
face of the membrana tympani. Finally divide the petrous 
bone with a fine hair-saw by an incision starting in at the 
styloid process and coming out at the carotid canal, parallel 
to the crest of the pyramid of the petrous bone. 

This incision divides the cavum tympani into halves. In 
the lateral half can be seen the membrana tympani with the 
hammer and the anterior half of the mastoid cells. In the 



POST-MORTEM EXAMINATIONS. 519 

median half are the labyrinthine wall of the cavum tympani 
with the stapes and the posterior half of the mastoid cells. 
It is best to remove the anvil before sawing through the 
bone. The Eustachian tube can be easily exposed by start- 
ing from its termination in the middle ear. 

The Naso -pharynx. — Although a fair view of the nares 
and pharynx can be obtained by chiselling off the portion 
of the base of the skull lying over them, the method does 
not begin to offer the satisfactory view that can be obtained 
by the method of Harke, 1 a method which is not so difficult 
as might at first sight seem, and which consists in halving the 
base of the skull by a longitudinal incision. To do this the 
original incision in the scalp must be extended on each side 
over the mastoid processes and along the anterior edge of 
the trapezius muscle to a point below the middle of the neck. 
Then the posterior flap and the underlying muscles must be 
freed from the occipital bone and the upper portion of the 
cervical vertebrae. In like manner, the anterior flap must 
be dissected from over the root of the nose and the upper 
edge of the orbits, and be drawn down over the face. Then 
flex the head strongly forward and saw through the occipital 
bone and the base of the skull, dividing the occipital and 
frontal bones, the sella turcica, the cribriform plate, and the 
basilar process into equal halves. Anteriorly, it is well to 
go a little to the left or right, so as not to injure the nasal 
septum. 

The next step is to cut the pachymeninx and the appara- 
tus ligamentosis between the anterior edge of the foramen 
occipitale magnum and the processus odontoideus, as well 
as the inner side of the atlanto-occipital joint from within. 
Then the two halves of the skull are to be drawn forcibly 
apart. The nasal bones, the hard palate, and the alveolar 
process of the upper jaw break, and the two halves of the 
base of the skull open like a book, revolving around an axis 
which passes through the joint of the lower jaw and the 
atlanto-occipital joint. 

If the foramen occipitale magnum offer too much resist- 

1 Berliner klin. Wochenschrift, 1892, No. 30. 



520 PATHOLOGICAL TECHNIQUE. 

ance, break through it with a chisel, and also if necessary 
through the anterior and posterior arches of the atlas. 

It is now easily possible to inspect the sinus sphenoidales, 
the nasal septum, the frontal sinuses, and the nasal passages. 
The antrum of Highmore is easily opened with forceps and 
a pair of bone-shears. 

After the operation the two halves of the base of the skull 
are brought together, and wired if necessary. When the 
skin-flaps have been replaced all evidence of the operation is 
covered up. 

Examination of New-born and Very Young Chil- 
dren. — i. The head is preferably opened by the method given 
on page 507. 

2. According to Nauwerck, the spinal canal can be opened 
by dividing the vertebral arches with strong scissors. 

3. The umbilical cord, if present, and the umbilical arte- 
ries demand close attention in children who have lived a few 
days or weeks, for the purpose of determining if infection has 
taken place at that point. Nauwerck advises a modification 
of the primary long incision. A little above the umbilicus 
it should divide into two diverging incisions running to the 
pubes. In this way a triangular flap is left containing the 
umbilical arteries, while from the upper end is given off the 
umbilical vein. The vessels may be ligated or opened at 
any point that seems advisable. 

4. Anomalies of circulation should be looked for in all 
" blue babies." The closure or non-closure of the ductus 
Botalli (arteriosus) is best determined in situ by dissecting 
off the thymus and opening up the pulmonary vein in the 
middle of its anterior surface. The cut may be extended 
downward, if desired, through the pulmonary valve and the 
wall of the right ventricle. The duct lies in the median line 
of the pulmonary artery, a little above its division into its 
two main branches. A small probe can be passed through 
it into the aorta. The condition of the foramen ovale be- 
tween the auricles is easily examined. 

For other anomalies of the circulation it will usually be 
found most satisfactory to remove the thoracic organs in 



POST-MORTEM EXAMINATIONS. 52I 

mass, so as to be able to open up the heart and the vessels 
given off from it before any of the vessels have been severed 
from their connections. 

5. In medico-legal cases especially it is important to de- 
termine whether or not a child has breathed. The main 
steps of the process are as follows : 

(a) Position of the diaphragm before the chest is opened. 
When the lungs are fully distended it is at the fifth or sixth 
rib on the right and at the sixth rib on the left. When the 
lungs contain no air or are but partially distended the 
diaphragm reaches to the fourth rib. 

(b) Ligate the trachea above the sternum before opening 
the thorax. 

(c) After examining the heart, etc., divide the trachea above 
the ligature and remove the thoracic organs in one piece. 

(d) Dissect off the thymus gland and the heart, and place 
the lungs in a large dish of clear cold water to see if they 
will float or not. 

(e) Incise the lungs and notice if they crepitate ; squeeze 
the lung-tissue gently, and see if bubbles of air mingle with 
the blood on the surface, or squeeze the lung beneath water 
and observe if bubbles of air rise to the surface. Decompo- 
sition may give rise to gas in the lungs. 

(/) Divide the lungs into lobes, and then into small 
pieces, and determine if any of them will float. 

Table of the Weight and Length of the Fetus at each Month 
of Gestation (from v. Hecker, cited by Nauwerck). 



Time in months. 


Weight. 


Leng 


2 


4gr- 


2-5-3 


3 


5-20 " 


7-9 


4 


I20 " 


IO-I7 


5 


284 « 


18-27 


6 


434 " 


28-34 


7 


1218 " 


35-38 


8 


1549 " 


39-41 


9 


1971 « 


42-44 


10 


2334 " 


45-47 



6. The long bones should be incised, so as to expose the 



5 22 PATHOLOGICAL TECHNIQUE. 

epiphyseal line, which should be examined for evidences of 
congenital syphilis. The ends of the femur and tibia at the 
knee are usually chosen. For making the incision a fine 
hair-saw is preferable to a knife, because the latter often 
causes the bone to break apart at the epiphyseal line. 

The age of the fetus in months can be determined after 
the fifth month by dividing the length in cm. by 5. 

Weight of Organs in a New-born Child. 

Brain 380 gr. (Bischoff). 

Thymus 14 « (Friedleben). 

Heart 20.6 " (Thoma). 

Lungs 58 " 

Spleen ii.i " 

Kidneys together 23.6 " (Thoma). 

Testicles 8 " 

Liver 118 " 

Restitution of the Body. — After an autopsy is finished 
it is necessary to put the body into such a condition that no 
evidence of the operation will be noticed except on careful 
inspection. All fluids should be removed from the cavities. 
Organs not required for further examination should be re- 
placed. The brain is placed in the body-cavity because it is 
usually impossible to restore it to the skull. The best mate- 
rial for filling up the cavities is fine sawdust. It packs easily 
and smoothly, absorbs well, keeps the needle dry so that it 
does not slip, and does not interfere with sewing like oakum, 
which gets into the stitches. In private autopsies any make- 
shift, such as bran, newspapers, or cloth, must be employed. 
If the pelvic organs have been removed, stuff the pelvis 
tightly to prevent leakage. The cranium may be left empty, 
although it is usually better to pack a little sawdust or other 
material into the base of the skull and the upper part of the 
spinal canal to prevent leaking. Sometimes it is advisable to 
fill the cranial cavity with sand or sawdust wrapped tightly 
in a cloth, of which the edges are brought together and 
twisted so as to crowd the material into a compact mass. If 
the thoracic cavity is well packed with sawdust, the sternum 
will stay perfectly in place without being sewed. 



POST-MORTEM EXAMINATIONS. 523 

If part of the vertebral column has been removed, a stick 
or heavy iron rod should be run into the spinal canal above 
and below, so as to stiffen the body and hold it in position 
while it is filled about half full of plaster of Paris. After 
this has set there is little danger of the body losing its form. 

In sewing up the body-cavity, begin at the neck. Use a 
piece of twine a little over one and a half times the length 
of the incision. Take one stitch and fasten the end with a 
simple knot or with a surgeon's knot. Turn the loose end 
in under the skin. Hold the attached end of the twine taut 
with the left hand about 8 to 10 cm. from the line of incis- 
ion. The needle is then passed from within outward through 
the edge of the flap and in a diagonal line from below up- 
ward. The stitches should be from 1 to 2 cm. apart, and 
about the same distance from the edge of the flap. The ob- 
ject of keeping the end of the twine taut is to keep the 
sutures tight and the edges of the flaps up so that the needle 
can be thrust in easily. 

Arrived at the lower end of the incision, take two button- 
hole stitches and draw them tight. Then take a long stitch 
off to one side and cut the twine close to the skin, so as to 
bury the end of it deeply and securely. 

If in removing the calvarium the precaution is taken to 
crack at least a part of the inner table with the chisel and 
hammer, projecting pieces of bone are usually left which 
interlock and hold the calvarium snugly in position when 
it is replaced. It is further fastened by sutures on each side 
through the fascia of the temporal muscle. It is always 
more difficult to sew up the incision in the scalp than the 
one in the body, especially when the hair is long. Care 
should be taken to bury the ends of the suture securely. 

The skull of a child is so thin that it is usually best to 
wire the calvarium in place or fasten it by means of double 
tacks, otherwise it may slip out of place after the scalp has 
been sewed up. 

Slee's ingenious method deserves mention. The usual 
saw-cuts in the skull over the ear are allowed to cross each 
other, so that slits about an inch long are formed in the tern- 



524 PATHOLOGICAL TECHNIQUE. 

poral bone. An ordinary roller bandage is stretched across 
the skull and crowded edgewise into the slits. Then the cal- 
varium is replaced and the ends of the bandage are tightly 
overlapped over the vertex and secured by pins. 

List of Publications on Post-mortem Technique. 

1. Virchow, Sektionstechnik, 4 Aufl., 1893. 

2. Orth, Pathologish-anatomische Diagnostik, VI Aufl., 1900. 

3. C. Nauwerck, Sektionstechnick fur Studierende und Aerzte, V Aufl., 
Jena, G. Fischer, 191 2. 

4. Chiari, Pathologisch-anatomische Sektionstechnik, Berlin, II Aufl., 1907. 

5. G. Hauser, Die Zenkersche Sektionstechnik, Jena, G. Fischer, 1913. 



ADDENDA. 



Method of Preparing the " Bacterial Vaccines" 
of Sir A. 1$, Wright. — These " vaccines" are suspensions 
of definite quantities of bacteria killed by heat, in a 0.9 per 
cent, solution of sodium chlorid. The method here described 
is a modification of Sir A. E. Wright's method as used in 
the Pathological Laboratory of the Massachusetts General 
Hospital. 

As profuse a growth as possible of the bacterium is ob- 
tained in a number of " slant " culture-tubes, three or four 
tubes usually furnishing a sufficient mass of bacteria for the 
purpose. All of the bacterial growth in the tubes is col- 
lected in a thick suspension in sterile 0.9 per cent, saline 
solution in a sterile test-tube. This test-tube is then drawn 
out with the aid of a blast-lamp to a small diameter some centi- 
meters above the level of the fluid, and is set aside to cool. 
When cool, the drawn-out portion is sealed off in the flame 
and the tube is thoroughly shaken during some minutes. 
The sealed extremity is then opened and a few drops of the 
suspension withdrawn into a small dish or onto a block of 
paraffin for the purpose of later determining the number of 
bacteria in suspension, after which the tube is again sealed in 
the flame. The suspension is now ready for sterilization. 
This is done by keeping the tube fully submerged in a water- 
bath at 6o° C. for from one and a half to two hours. 

The determination of the number of bacteria per cubic 
centimeter in the sample withdrawn from the sealed tube is 
made as follows : 

The first step in the process is to thoroughly break up the 
clumps of bacteria so that each bacterium, as far as practic- 
able, is free and separate in the suspension. This may 
have been already accomplished by shaking the suspension 

525 



526 PATHOLOGICAL TECHNIQUE. 

in the test-tube, but if not, then the breaking up of the 
clumps may be effected with the aid of a capillary pipette 
about I mm. in diameter, prepared from a piece of glass 
tubing of about the diameter used for the ordinary medicine 
dropper. To this pipette is affixed a tightly fitting rubber 
bulb similar to that used on a medicine dropper, but of the 
best quality of rubber. The smaller end of the pipette must 
be squarely broken off. The breaking up of the clumps is 
effected by repeatedly forcing the bacterial suspension in and 
out of the pipette by manipulation of the bulb, while the 
pipette is held perpendicularly against the surface of the 
glass dish or paraffin block in such a way as to bring as 
much as possible of the circumference of the smaller end in 
contact with it, thus leaving minute clefts which are small 
enough to cause the breaking up of the clumps as they are 
forced through. In this process the pipette is most conve- 
niently held in such a manner that the bulb may be manipu- 
lated with the thumb and forefinger, while the remaining 
fingers grasp the body of the pipette and steady it against 
the surface of the glass dish or paraffin block. With some 
bacteria, for example, the gonococcus, this procedure is not 
sufficient to break up the clumps, and in this case the shak- 
ing of some cubic centimeters of the suspension with fine 
sterilized sand in a small tube is resorted to. 

The next step is to determine the number of bacteria per 
cubic centimeter in the suspension. This may be done in 
either of two ways. 

One way is to mix thoroughly equal quantities of freshly 
drawn normal blood, of a fluid which prevents the coagula- 
tion of the blood, and of the suspension ; then in stained 
smear preparations of the mixture determine the ratio be- 
tween the number of red blood-corpuscles and the number 
of bacteria. Assuming five million red blood-corpuscles to a 
cubic millimeter, the number of bacteria per cubic centimeter 
is readily determined. 

This procedure is carried out with the aid of a capillary 
pipette provided with a rubber bulb like the pipette described 
above. The mark is made on the pipette 2 or 3 centimeters 



ADDENDA. $2 J 

from its smaller extremity, and into the pipette, while grasped 
in the hand, as before described, there is drawn up to this 
mark successively the fresh blood, the anti-coagulating fluid, 
and, finally, the bacterial emulsion, a small amount of air 
beine allowed to enter the tube after each measure of fluid, 
and the end of the pipette wiped after each taking - . The 
contents of the pipette are immediately expressed onto a 
glass dish or paraffin block, and the elements in the various 
fluids thoroughly mixed by drawing the mixture in and out 
of the pipette repeatedly. Smears are then prepared and 
stained with Wright's blood-stain, as in the case of blood- 
smears. The counting is done under an oil-immersion objec- 
tive with an eye-piece, upon the inferior lens of which a 
square has been marked out about 9 mm. on a side, with a 
wax pencil. The number of red blood-cells and bacteria 
seen within this ruled square are counted in various portions 
of the preparation until IOOO red cells have been counted. 
The anti-coagulating fluid employed consists of 1.5 per cent, 
sodium citrate in 0.9 per cent, sodium chlorid solution. 

Another way of determining the number of bacteria per 
cubic centimeter in the emulsion is to count the bacteria 
without staining in a chamber similar to the Thoma-Zeiss 
blood-counting chamber. This method has been devised by 
one of us, and is regarded as much easier of execution than 
the one above described. The chamber used is manufactured 
by Zeiss for counting blood-plates by the Helber method. 
It should be supplied with an especially thin cover-glass 
(No. 146, Zeiss' Catalogue) to permit the use of the high- 
power dry objective with which the counting is made. The 
chamber is ruled like the " Thoma-Zeiss blood-countingr 
chamber," and the rulings have the same value, except that 
the chamber is 0.02 mm. deep instead of 0.1 mm. For 
counting, the suspension of bacteria is diluted and mixed 
with distilled water 1 : 200 with the aid of the red blood- 
corpuscle pipette of the " Thoma-Zeiss " apparatus. By a 
simple calculation it will be apparent that the product of the 
multiplication of the average number of bacteria per small 
square by 4000 million will be the number of bacteria per 
cubic centimeter. 



528 PATHOLOGICAL TECHNIQUE. 

When the heating of the suspension is finished, the small 
end of the sealed tube is broken and a "planting" made from 
the emulsion upon the surface of a blood-serum "slant" to 
test the sterility of the emulsion. Immediately after this a 
sufficient quantity of the emulsion is mixed with sterile 0.9 
per cent, saline solution to give a dilute suspension of the 
volume of 50 c.c. containing the required number of bacteria 
per cubic centimeter. This is done as follows: 

A small flask containing 50 c.c. of 0.9 per cent, of saline 
solution, closed with a rubber nipple, and the whole sterilized, 
is previously prepared. The quantity of the suspension neces- 
sary to give the desired number of millions of bacteria per 
cubic centimeter in a volume of 50 c.c. of saline solution hav- 
ing been determined by calculation, this quantity is withdrawn 
from the flask by means of the sterilized hypodermic syringe, 
the needle of the syringe being plunged through the rubber 
nipple while the flask is inverted ; then the calculated quan- 
tity of the suspension is drawn up into the syringe and in- 
jected into the saline solution by passing the needle through 
the rubber nipple as before. Following this, o. 1 5 c.c. of lysol 
is similarly injected into the flask through the rubber nipple, 
and, after shaking, this diluted suspension constitutes the 
vaccine. Before injecting it, it should have been proved 
sterile. 

The vaccines prepared from the staphylococci are made 
up so as to contain 600 million staphylococci in each cubic 
centimeter, while those of other bacteria are made up to 
contain only 100 million. 

The dose varies according to the circumstances of the 
case. The full dose of the staphylococcus vaccine is 600 
million, while a full dose of other vaccines is 100 million. 
The injection is made subcutaneously, usually in the abdom- 
inal wall in men and between the shoulder-blades in women, 
these being readily accessible and less sensitive areas. In 
charging the syringe for the injection, the needle is passed 
through the rubber nipple with the flask inverted. Before 
doing this, the surface of the rubber nipple should be steril- 
ized, either with lysol, or by plunging the nipple and neck 
of the flask into hot water for a few seconds. 



529 



ADDENDA. 

Methyl-violet Shellac. — 

Best white shellac, 10 gm. ; 

Alcohol, 95 per cent., 20 to 25 c.c. ; 

Methyl-violet, 0.1 gm. 

This solution will be found very convenient for marking 
important fields in mounted sections. It may be used with 
the circular markers made for this purpose, but a pen is just 
as convenient and less liable to cause injury to the prepara- 
tion by pressure. The desired field is readily outlined under 
the low power of the microscope by a series of dots or a 
continuous line. The solution after drying is insoluble in 
xylol or water. 

METHOD FOR BLACKENING TABLE TOPS. 

Solution I. 
125 grams copper sulphate ^) 
125 grams potassium chlorate VBoil until dissolved. 
1000 c.c. water ) 

Solution II. 
150 grams anilin hydrochlorate, 
1000 c.c. water. 

Use white wood for tops, since it is cheaper and takes stain 
better than pine. Oak gives good results. 

Spread papers to cover floor completely under and around 
tables. 

1. Apply with brush two coats of Solution I while hot ; 
the second as soon as the first is dry. 

2. When second is dry, apply in the same way two coats 
of Solution II and let them dry thoroughly. 

3. Put on with a cloth thin coat of raw linseed oil and 
polish thoroughly by rubbing. 

4. When dry wash thoroughly with hot soapsuds. 

5. Rub again with raw oil. 

6. Rub with cotton waste over and over again until the 
black does not come off. This requires hard work. . 

To keep tops in perfect condition, rub off once a week 
with white (liquid) petrolatum. 

34 



530 PATHOLOGICAL TECHNIQUE, 

N. B. — If table tops are blotched with paraffin, wax, or 
resin, remove these before beginning to paint. Stains do 
not harm. Paint top surface and edges only. 

TO CLEAN SLIDES. 

Slides and cover-slips are cleaned by dipping in alcohol and 
wiping dry with a soft crash towel or old linen handkerchief. 

Cover-slips, after they are clean, should be preserved dry 
in covered dishes. The common method of keeping them 
under alcohol cannot be recommended. 

To clean old slide preparations, heat them until the bal- 
sam softens so that cover-slips and slides can be drawn 
apart. The slides and cover-slips are then treated sepa- 
rately, either by boiling them for an hour or so in a strong 
solution of powdered soap followed by water and then acid 
alcohol or by placing them in nitric acid. A 10 per cent, 
solution is usually sufficient, but occasionally the strong acid 
will be found necessary. Some workers prefer equal parts 
of alcohol and hydrochloric acid; others, a 5 per cent, solu- 
tion of glacial acetic acid in 95 per cent, alcohol; still others, 
the following mixture: 

Bichromate of potassium, 2 parts; 

Sulphuric acid, 3 

Water, 25 "' 

A thorough washing in running water, followed by alco- 
hol, completes the process. 

Hand Lotion. 

Gum tragacanth, 9 to 10 gm. ; 

Boric acid, 20 " 

. Glycerin (double distilled), 50 c.c. ; 

Water, ad. 1000 " 

Oil of rose geranium, 1 

Mix the first four ingredients in a bottle, and place in a 
warm place. Shake occasionally. After one to four days 
(whenever the gum is thoroughly softened) filter or squeeze 
through fine cheese-cloth or a towel, on the dry surface of 
which the oil of geranium has been poured. The amount 
of glycerin can be increased if desired. 



INDEX 



Abbe camera lucida, 20 

illuminating apparatus, 19 
Abbott's method for staining spores, 

238 
Abdomen, incision to open, 483 
Abdominal cavity, inspection of, 484 
opening of, 483 
organs, removal of, 495 
Acetic acid for fat, 166 
for fresh tissue, 34 
Acetic-osmic bichromate fixation of 

mitochondria, 107 
Acid alcohol, 92 
method, 92 
fuchsin, 79 

and hematoxylin, Pianese's, 83 
and picro-nigrosin, Pianese's, 82 
malachite-green, and Martius 
yellow, Pianese's, 82 
and nigrosin, Pianese's, 81 
Mallory's, 115 
hematoxylin, phosphomolybdic, 70 

phosphotungstic, 71 
osmic, as fixative, 48 
polychrome methylene-blue solu- 
tion, Goodpasture's 75, 156 
Actinomyces bovis, 362 
clubs, 363-368, 370 
cultures, 367 
diagnosis, 366 
isolation, 368 
Mallory's stain for, 372 
pathogenesis, 368 
staining in sections, 371 
Actinomycosis, 362 

micro-organism of, 362 
Adrenals, removal of, 500 
Agar, blood, preparation, 197 
Agar-agar, glucose, preparation, 196 
glycerin, preparation, 196 
hydrocele fluid, 273 
lactose-litmus, preparation, 196 
mannite litmus, 301 
plain, precipitates in, 195 
preparation, 193 
Agglutination reaction of comma 

bacillus, 359 
Albumin in hydrothorax, 455 



Albuminous degenerations, 164 
Alcohol, acid, 92 

and formaldehyde as fixative, 43 

as fixative, 39 

hematoxylin, Weigert's, 70 

one-third, Ranvier's, 35 
Alexine, 427 
Algeri's stains for fatty degeneration 

of nervous tissue, 147 
Alkaline methylene-blue, Unna's, 74 
Alphanaphthol-pyronin stain, Gra- 
ham's, for oxydase granules, 423 
Altmann's aniline acid fuchsin solu- 
tion, 79 
Alum carmine, 71 
method, 92 

cochineal, 72 
method, 92 
Alum-hematein, glycerin, Mayer's, 

70 
Alum-hematoxylin, 67 

aqueous, method, 88 

Harris', 69, 88 

Mallory's, 68, 88 
Alzheimer's method for cytological 

examination of cerebrospinal fluid, 

449 
Amboceptors, bacteriolytic, 428 
hemolytic, 428 
in serum diagnosis of syphilis, 428, 

433 
antihuman, 433 
antisheep, 435 
Noguchi's standardization, 

437 
Wassermann's standardiza- 
tion, 437 
Ameboid movements of Plasmodium 

malaria?, 385, 386 
Amoeba, 381. See also Entamceba. 
Amputating-knives, 474 
Amyloid infiltration, 180 

Bismarck-brown for, 182 
iodin for, 180 

and sulphuric acid for, 181 
iodin-green for, 182 
Langhans' method for permanent 
mounts, 181 

53i 



532 



INDEX. 



Amyloid infiltration, Mayer's stain 
for, 182 
methyl- violet for, 181 
stains for, 180 
Anaerobes, cultivation, 220 
Buchner's method, 223 
Liborius' method, 221 
plate-cultures, 223 
Wright's method, 225 
Zinsser's method, 224 
culture-media for, 221 
Anesthetizing rabbits, 230 
Aniline acid-fuchsin-solution, Alt- 
mann's, 79 
and xylol as clearing reagent, 105 
blue for nerve-fibers, Stroebe's, 134 
for nervous system, 1 24 
Mallory's, for connective tissue, 
j ' in 
dyes, 73 

for bacteria in sections of tissue, 

246 
for mucin, 172 
oil as clearing reagent, 104 
safranin, Babes', 78 
stains, 93 
Aniline-fuchsin, 76 
Animal parasites, 381 

examinations for, 385 
in sputum, 237, 466 
Animals, care of, 233 
food for, 233 
inoculation of, 229 
in diagnosis, 461 
quantity of bacteria used, 232 
Anthrax bacillus, 327 

diagnosis, 330 
Antibodies, 429 

Antiformin method for cultivating 
tubercle bacilli in sputum, 346, 

349 . 
for obtaining pure cultures of 
tubercle bacillus, 346 
Antigen in serum diagnosis of syph- 
ilis, 429, 435 
Noguchi's standardization, 

439 
Wassermann's standardiza- 
s tion, 440 

Aorta, opening of, 505 
Aqueous alum-hematoxylin, 67 

method, 88 
Artificial serum, 35 
Ascites dextrose bouillon, 258 
Asiatic cholera, spirillum of, 355. See 

also Comma bacillus. 
Auricles, opening of, 488 
Autochthonous pigments, 183 
Autoclave for sterilization, 207 
Autopsy, 472 



Autopsy, cultures at, 214 

finger cuts during, 478 

general rules for, 477 

instruments for, 473 

operator's hands after, 478 

preparations, 477 

private, 479 

record of, 477 

restitution of body after, 522 

room for, 477 

rubber gloves for, 478 

suggestions to beginners, 478 

wounds during, treatment, 478 
Autopsy-bag, 479 
Autopsy-knife, 474 
Autopsy-needles, 476 
Autopsy-table, 473 
Axis-cylinder processes, stains for, 

125, 129 
Axis-cylinders of nerve-fibers, gold 

stain for, 101 



Babe's aniline safranin, 78 
Bacillus abdominalis, 293. See also 
Bacillus typhosus. 
aerogenes capsulatus, 334 
Bordet-Gengou, 286 
coli communis, 288 

bacillus typhosus and, differ- 
entiation, 299 
colon group, 291 
comma, 355. See also Comma 

bacillus. 
diphtheriae, 304 
diagnosis, 308 
staining, 309 

Hunt's method, 311 
Mallory's method, 311 
toxin-production of, 307 
dysenteriae, 300 
lactis aerogenes, 291 
Loffier's, 304 
staining, 309 

Hunt's method, 311 
toxin-production, 307 
mallei, 314 

diagnosis, 317 
mucosus capsulatus, 331 
of anthrax, 327 
diagnosis, 330 
of bubonic plague, 323 

diagnosis, 327 
of chancroid, 319 
of Ducrey, 319 
of glanders, 314. See also Glanders 

bacillus. 
of green pus, 321 
of influenza, 282 
diagnosis, 285 



INDEX. 



533 



Bacillus of influenza, staining, in sec- 
tions, 286 

of Koch, 342. See also Bacillus 
tuberculosis. 

of leprosy, 353. See also Leprosy, 
bacillus of. 

of malignant edema, 340 

of rhinoscleroma, staining, 334 

of syphilis, staining, 397 

of tetanus, 336 
toxin of, 339 

of typhoid fever, 293. See also 
Bacillus typhosus. 

of whooping-cough, 286 

paratyphoid, 299 

perfringens, 334 

pertussis, 286 

pneumoniae of Friedlander, 332 

proteus, 304 

pyocyaneus, 321 

pyogenes fcetidus, 291 

scarlatinas, 312 

tuberculosis, 342 

antiformin method for obtaining 
pure cultures of, 346, 349 
bovine and human, 345 
cultivation of, from sputum 
and feces, Petroff's method, 

345 
diagnosis, 347 
Ehrlich's stain for, 352 
in celloidin sections, staining, 

353 
in feces, 350 
in pus, 350 
in sputum, 466 

antiformin method of cultivat- 
ing, 346, 349 

examination for, 348 
in tissues, 350 
in urine, 350 
inoculation of guinea-pigs with, 

isolation of, 344 

Kiihne's method for, 352 

leprosy bacillus and, differentia- 
tion, 348 

smegma bacillus and, differentia- 
tion, 348 

staining, 351 

Ziehl-Neelson-Gabbet stain for, 
352 
typhosus, 293 

bacillus coli communis and, dif- 
ferentiation, 299 

blood-serum reaction, Widal, 297 

clump reaction, 300 

diagnosis, 299 

examination of feces for, 302 

from blood during life, 298 



Bacillus typhosus, ox-bile method of 
cultivating, 298 

serum reaction, 300 

staining, 296, 297 

Widal reaction, 297 
Welchii, 334 
Bacteria, anaerobic, cultivation, 220 

See also Anaerobes, cultivation: 
capsules of, Hiss' method of stamp- 
ing, smears, 245 : 

Muir's method of staining, 
smears, 246 

Smith's method of staining, 
smears, 244 
decolorized by Gram's method, 237 
development of, hanging-block 

method of observing, 228 
Gram-Weigert method for, 250 
in sections, analine dyes for, 246 

embedding, 247 

Giemsa's stain, 392 

Lofner's methylene-blue for, 248 

staining, 246 '; 

Unna-Pappenheim's methyl- 

green-pyronin stain for, 249 : 

in smear preparations, staining of, 

234 
in sputum, Smith's stain for, 237, 

466 
injection of, into mesenteric veins, 

230 
motility of, determination, 227 
not stained by Gram, 248 
pathogenic, 253 
putrefactive, 304 
quantity of, used in inoculation, 

232 : 

skin, examination for, 160 1 

stained by Gram's method, 237, 
249 

by tubercle bacillus method, 

35i 
by Verhoeff's modified Gram 
stain, 250 
staining of, methods, 234 
Bacterial vaccines of Wright, prepa- 
ration, 525 
Bacteriologic examination, 209 

material for, collection of, 209 
Bacteriolysins, 428 
Bacteriolytic amboceptors, 428 
Bacterium, isolation of, in pure cul- 
ture from mixed growth, 217 • 
Balsam, Canada, 106 1 

chloroform, 106 
neutral, 105 
xylol, 106 
Band-saw, 474 

Bath, water-, in serum diagnosis of 
syphilis, 433 



534 



INDEX. 



Baumgarten's stain for leprosy bacil- 
lus, 354 
Benda's stain for fat acid crystals, 168 
for myoglia fibrils, 120 
for neuroglia fibrils, 146 
Benian's stain for treponema palli- 
dum, 399 
Bensley's stain for mitochondria, 107 
Benzene in paraffin embedding, 60 
Benzol in paraffin embedding, 60 
Bergamot oil as clearing reagent, 104 
Berlin blue as injection-mass, 37 
Best's carmine for glycogen, 178 
Bethe's method of fixing methylene- 

blue for nerve-fibers, 131 

Bielschowsky's method of staining 

axis-cylinders and neurofibrils by 

silver impregnation, 132 

silver stain for collagen fibrils, 113 

Bile-pigment, bilirubin-hematoidin, 

183 . 
Bilharzia, 405 
Bilirubin, bile-pigment-, 183 

hematoidin-, 183 
Biondi-Heidenhain's stain, 79 

method, 96 
Bismarck-brown, 78 

for amyloid, 182 
Biuret test, 462 
Blackening table tops, method for, 

529 
Bladder, gall-, dropsy of, fluid of, 462 

opening of, 503 
Blastomycetes, 376 

diagnosis, 380 
Blastomycetic dermatitis, 376 
Blastomycosis, 376 
Blood, agar, preparation, 197 

bacillus typhosus from, during life, 

298 
collection of, for cultures, Mcjun- 

kin's tube for, 425 

corpuscles, counting of, 410 

red. See Red corpuscles. 

white. See Leucocytes. 

cover-glass preparations, 415 

cultures during life, 425 

of gonococcus, 276 
Ehrlich's triple stain for, 41 7 
examination of, 410 

without drying or fixation, 421 
in gastric contents, 469 
Leischmann's stain for, 418 
leucocytes of. See Leucocytes. 
malarial organisms in, examination, 

389 
red corpuscles. See Red corpuscles. 
smear preparations on slides, 417 
stains for, 417 
test for, 469 



Blood, white corpuscles of. See Leu- 
cocytes. 
Wright's stain for, 106, 418 

Blood-agar medium for comma bacil- 
lus, 362 

Blood-counting instrument, 410 

Blood-films, staining, 420 

with Wright's stain, microscop- 
ical appearances in, 420 

Blood-globules, red, in sputum, 465 
white in sputum, 465 

Blood-platelets, 421 

Wright's method of counting, 414 
stain for, 150 

Blood-serum, collection of, 198 
cultures on, preparation, 212 
preparation, 198 
reaction in typhoid, 297, 300 

Blood-vessels, opening of, 494 

Blue babies, 520 

Berlin, as injection-mass, 37 
coloring mass as injection, 36 

Boards for autopsy, 476 

Boas' resorcin test for hydrochloric 
acid, 470 

Body, external examination of, 481 
internal examination of, 482 
parts of, special inspection, 482 
restitution of, after autopsy, 522 

Body-length, measuring of, 481 

Boiling as fixative, 49 

Bolton's potato-cultures, preparation 
201 

Bone, 157 

cartilage and, differentiating, 158, 

i59 

saws, 474 

stains for, 157, 158, 159 
Bone-cutter, 476 
Bone-marrow, 149 

sections, 149 

smear preparations, 153 

staining, 149 
Borax methylene-blue, Sahli's, 75 
Bordet-Gengou bacillus, 286' 
Bothriocephalus latus, 405 
Bottles, dropping, 31 

for histologic work, 31 
Bouillon, dextrose, ascites, 258 
preparation, 192 

glucose, preparation, 192 

hydrocele, 276 

preparation, 190 
Bovine tubercle bacillus, human and, 

345 
Brain, examination of, 510 
frontal section, 515 
occipital section, 515 
parietal section, 515 
pediculo-frontal section, 515 



INDEX. 



535 



Brain, pediculo-parietal section, 515 
prefrontal section, 515 
removal of, 505, 508 
section of, 511 

Pitre's method, 514 
Virchow's method, 513 
weight of, 510 

Bronchi, opening of, 494 

Bronchioles, fibrinous casts of, in 
sputum, 464 

Brown and Smith's method of culti- 
vating tubercle bacilli from spu- 
tum, 346 

Bubonic plague, bacillus of, 323 
diagnosis, 327 

Buchner's method for anaerobes, 
223 

Burri's stain for treponema pallidum, 
401 

Cajal's double method for nervous 

tissue, 128 
Calcification, stains for, 187 
Calcium deposits, stains for, 187 
Calvarium, removal of, 507 

Slee's method of restitution, 523 
Camera lucida, Abbe, 20 
Canada balsam, 106 
Capsule of pneumococcus, 265 

staining, 237 
Capsules in sections, staining, 251 
Smith's method, 251 
of bacteria, Hiss's method for 
smears, 245 
Muir's method for smears, 246 
Smith's method for smears, 244 
Carbohydrates, fermentation of, 259 
Carbol-fuchsin solution, Verhoeff's, 

75 
Ziehl-Neelson's, 75 
Carbol-gentian- violet, 77 
Carbolic acid and xylol as clearing 

reagent, 105 
Carbon as pigment, 184 
dioxid in freezing, 20 
Carmine, alcoholic, Mayer's, 72 
alum, 71 

method, 92 
and picro-nigrosin, Pianese's, 80 
injection-mass, 36 
lithium, method, 92 

Orth's, 72 
neutral, 72, 96 
stains, 71, 92 
Best's, for glycogen, 178 
method, 92 
Carmine-gelatin as injection-mass, 37 
Cartilage, 157 
bone and, differentiating, 157, 158, 
iS9 



Cartilage, stains for, 157, 158, 159 

Cartilage-knives, 474 

Caseation, 170 

Caseous masses in sputum, 464 

Casts of bronchioles, fibrinous, in 

sputum, 464 
Caustic potash as macerating fluid, 

35 
Cedar, oil of, as mounting reagent, 

106 
Cedar-wood oil as clearing reagent, 

104 
Celloidin, 56 
embedding, 55, 56 
method for serial sections, 63 
microtome, 22 
Schering's, 56 

sections attaching to slides, 58 
tubercle bacillus in, staining, 353 
vulcanized fibers for mounting, 
26 
Cells, mast-. See Mast-cells. 
plasma, cytoplasm of, Schridde's 
method for demonstrating gran- 
ules in, no 
Cellulose, iodin and sulphuric acid 
for, 181 
stains for, 181 
Central nervous system, aniline blue 
for, 124 
Cajal's double method for, 1 28 
chrome salts for fixing, 120 
Cox's modification of Golgi's 

stain for, 129 
degenerations of, stains for, 

146 
fixing reagents for, 120 
formaldehyde as fixative, 121 
Golgi's method for, 125-129 
Cox's modification, 129 
Kallius' fixing method, 
128 
Kallius' method for fixing Gol- 
gi's stains, 128 
Lenhossek's stain for, 125 
Nissl's stain for, 124 
phosphomolybdic-acid hema- 
toxylin for, 123 
phosphotungstic acid hema- 
toxylin for, 1 23 
stains for, 120 
general, 122 
syphilis of, Lange's colloidal 
gold test of cerebrospinal 
fluid for, 451 
treatment of sections, 128 
van Gieson's stain for, 122 
Centrifuge, 25 
in -serum diagnosis of syphilis, 
433 



53^ 



INDEX. 



Cerebrospinal fluid, Alzheimer's 
method for cytological exam- 
ination of, 499 

examination of, 447 

globulin in, Noguchi's method, 

449 
Ross- Jones method, 449 
Lange's colloidal gold test of, 
for syphilis of central nervous 
system, 451 
leucocytes in, 448 
proteids in, increase test, 449 
meningitis, bacterium of, 279 
Chain formation, demonstration, 257 
Chancroid, bacillus of, 319 
Charcot-Leyden crystals in sputum, 

467 
Children, young, autopsy on, 520 
Chisel for autopsy, 476 

hatchet-, 476 
Chlorid of gold as stain, 101 
for nerve-fibers, 130 
of iron and dinitroresorcin for 
nerve-fibers, 134 
hematoxylin, Mallory's, 90 
Chloroform balsam, 106 

in paraffin embedding, 59 
Cholera, Asiatic, spirillum of, 355. 
See also Comma bacillus. 
vibrio, 355. See also Comma bacil- 
lus. 
Cholesterin crystals in sputum, 468 
iodin sulphuric acid for, 181 
stains for, 169, 181 
Chrome salts as fixative, 46 

for central nervous system, 120 
Chromic acid as macerating fluid, 35 
Circular incision for opening skull, 

506 
Circulation, anomalies of, 520 
Claudius' method for staining fla- 

gella, 243 
Clearing reagents, 103 

aniline and xylol as, 105 

oil as, 104 
carbolic acid and xylol as, 105 
Dunham's mixture as, 105 
oil of bergamot as, 104 
of cedar-wood as, 104 
of cloves as, 104 

and thyme as, 104 
of lavendar as, 104 
of thyme as, 104 
and cloves as, 105 
oleum origani cretici as, 104 
Weigert's mixtures as, 105 
xylol as, 104 
Clegg and Musgrave's method of cul- 
tivating amoeba, 384 
Clinical pathology, 410 



Cloudy swelling, 164 
Cloves, oil of, as clearing reagent, 104 
and thyme as clearing reagent, 
104 
Clubs of ray-fungus, 363-368, 370 
Clump-reaction of comma bacillus, 

359 
of typhoid bacillus, 300 
Coccidioidal granuloma, 376 
Cochineal, alum, 72 

method, 92 
Collagen fibrils, 113 
Colloid, 174 
stains for, 1 74 
Unna's, 176 
Colloidal gold test, Lange's, of cere- 
brospinal fluid, for syphilis of 
central nervous system, 451 
Colon bacillus. See also Bacillus 
coli communis. 
group, 291 
Colonies, discrete, 216 

obtaining, 217 
Colophonium, 106 
Combination stains, 79 

method, 96 
Comma bacillus, 355 

agglutination reaction of, 359 

diagnosis, 361 

Dieudonne's blood-agar medium 

for, 362 
inoculation of guinea-pigs with, 

358 
Pfeiffer's reaction of, 359 
Complement fixation, 430 

in serum diagnosis of syphilis, 427, 

433 
Complement-fixation test in echino- 
coccus infection, 446 
in gonorrheal infections, 445 
Concretions in sputum, 465 
Congo-paper test for hydrochloric 

acid, 469 
Cork for mounting celloidin sections, 

26 
Cornea, silver staining of, 101 
Corpora amylacea, iodin and sul- 
phuric acid for, 181 
Corpuscles in blood, 410 
red. See Red corpuscles. 
white. See Leucocytes. 
Corrosive sublimate as fixative, 43 
Corrosive-sublimate-alcohol fixative, 

Giemsa's, 44 
Costotome, 476 
Cover-glass forceps, 234 
preparations, 234, 415 
fixing, 234 
from cultures, 234 
microscopic examination, 235 



INDEX. 



537 



Cover-glass preparations of blood, 

4i5 
staining, 234, 235. See also 
Stains for bacteria in smear 
preparations. 
Cover-slips, 29 
cleaning, 29 
Cox's modification of Golgi's corro- 
sive-sublimate method, 129 
Croupous pneumonia, bacterium of, 

263 
Crystallizing dishes, 30 
Crystals in sputum, 467, 468 
Culture methods, 209 

collecting material for, 209 
Culture-media, 169 

autoclave for sterilization, 207 
Dorset's egg, 344 
for anaerobes, 221 
for gonococcus, 273 
for ringworms, 162 
inoculation, 216 
preparation, 190 
quantity in test-tubes, 205 
reaction of, titration in adjustment, 

202 
Sabouraud's, for ringworms, 162 
spores in, destroying, 206 
sterilization, 206 
storage of, 208 

test-tubes for, preparation, 189 
Cultures, anaerobic, cultivation, 220. 
See also Anaerobes, cultivation. 
at autopsy, 214 
blood, during life, 425 
cover-glass preparations from, 234 

staining, 234, 235 
examination by, 212 
on blood-serum, 212 
plate, anaerobic, 223 
pure, isolation of bacterium in, 
from mixed growth, 217 
obtaining, 216 
slant, 194, 198, 201, 206 
stab, 196, 205 

deep, 223 
transplanting, 216 
Curschmann's spirals in sputum, 

464 
Cysticerci, 405 

Cysts, echinococcus, fluid of, 463 
ovarian, fluids of, 461 
pancreatic, fluids of, 462 
parovarian, fluids of, 461 
renal, fluid of, 462 
Cytodiagnosis, 456, 457 

method, 458 
Cytological examination of cerebro- 
spinal fluid, Alzheimer's method, 
499 



Cytoplasm of plasma-cells and lym- 
phocytes, Schridde's method for 
demonstrating granules in, no 



Damar, 106 

Darkschewitsch's method for serial 

celloidin sections, 64 
Death, sudden, autopsy guides, 479 

opening heart in, 490 
Decalcification, 49 

nitric acid in, 50 

phloroglucin and nitric acid in, 51 

Schridde's solution, 51 

sulphurous acid in, 52 

trichloracetic acid in, 52 
Deep stab cultures, 223 
Degenerations, albuminous, 164 

of nervous system, staining, 146 
Delafield's alum-hematoxylin, 68 

method, 88 
Dendritic processes, stains for, 125 
Dermatitis, blastomycetic, 376 
Dextrose bouillon, ascites, 258 
preparation, 192 

gelatin, preparation, 198 
Diagnosis, examination of tissues in, 

454 
Dibothriocephalus latus, 405 
Dieudonne's blood-agar medium for 

comma bacillus, 362 
Differential stains, 86 
Diffuse stain, 78 

eosin as, method, 95 
method, 95 

neutral carmine as, 96 
picric acid as, method, 96 
van Gieson's, method, 96 
Diluting, 214 
Dimethyl-amido-azo-benzol test for 

hydrochloric acid, 470 
Dinitroresorcin and chlorid of iron for 

nerve-fibers, 134 
Diphtheria bacilli, 304 
Diplococcus intracellularis meningi- 
tidis, 279 
diagnosis, 282 
pneumoniae, 263 
diagnosis, 266 
Discolorations, postmortem, 481 
Discrete colonies, 216 
methods for, 217 
obtaining, 217 
Distomum haematobium, 405 
Dittrich's plugs in sputum, 465 
Dorset's egg medium, 344 
Drop-bottle on microtome, 24 
Dropping-bottle, 31 
Dropsy of gall-bladder, fluid of, 462 
Ducrey's bacillus, 319 



538 



INDEX, 



Ductus Botalli, non-closure of, 520 
Dunham's mixture as clearing re- 
agent, 105 

peptone solution, preparation, 202 
Duodenum, opening of, 498 

removal of, 497 
Dura, inspection of, 508 

removal of, 508 
Dysentery, bacillus of, 300 

entamoeba in, 381 

examination of feces for, 302 



Ear, inspection of, 518 
Echinococcus cysts, fluid of, 463 

infection, complement-fixation test 
in, 446 < 
Edema, malignant, bacillus of, 340 
Egg medium, Dorset's, 344 
Ehrlenmeyer flasks, 206 
Ehrlich's aniline-gentian- violet, 77 

method for fixing blood prepara- 
tions, 416 
for mast-cells, 108 
for tubercle bacillus, 352 

triple stain for blood, 41 7 
Ehrlich-Westphal method for mast- 
cells, 109 
Elastic fibers in sputum, 465 

staining, 116, 117 
Embedding, celloidin, 55, 56 

for bacteria in tissue, 247 

paraffin, 55, 59 

processes for frozen sections, 
Wright's, 54 
Embolism, autopsy guides, 478 
Embryo trichinellae, 409 
Endothelial cells, silver staining of, 

100 
Entamoeba, 381 

coli, 381 

cultivation of, 384 

examination for, 381 

histolytica, 381 

in dysentery, 381 

staining of, 382 
differential, 382 
Enterotome, 474 
Eosin, 78 

and methylene-blue in borax solu- 
tion, Pianese's, 80 

and methylene-blue, Mallory's 
method, 94 

as diffuse stain, method, 95 
Eosinophiles after Wright's stain, 420 
Epithelial cells in sputum, 465 
Erysipelas, 263 

bacterium of, 263 
Erythrocytes. See Red corpuscles. 
Esophagus, removal of, 498 



Estivo-autumnal parasite, 385 

cycle, 388 
Examination of cerebrospinal fluid, 

447 
Exner's stain for myelin-sheath, 140 
External examination of body, 481 
Extraneous pigments, 184 
Exudations, acute inflammatory, 148 

examination, 455 
Eye, removal of, 517 
Eyenes and Sternberg's method for 

staining treponema pallidum in 

sections, 402 



Farcy, bacillus of, 314 

diagnosis, 317 
Fat, 165 

acetic acid for, 166 

acid crystals, Benda's stain for, 168 

Klotz's stain for, 168 
osmium for, 166 
reactions of, 165 
Scharlach R. for, 165, 167 
stains for, 165 
Sudan III. for. 165 
tests for, 165 
Fatty degeneration of myelin-sheath, 
stains for, 147 
of nerve-fibers, stains for, 147 
detritus in sputum, 465 
Fatty-acid crystals in sputum, 469 
Feces, cultivation of tubercle bacilli 
from, PetrofT's method, 345 
examination of, 470 
for typhoid, paratyphoid, and 

dysentery bacilli, 302 
in hookworm disease, 408 
preparation of media, 302 
tubercle bacilli in, 350 
Fermentation of carbohydrates, 259 
Fermentation-tube, 292 
Fetus, age of, 522 

monthly length of, 521 
weight of, 521 
Fibers, elastic, in sputum, 465 

Weigert's stain for, Hart's mod- 
ification, 117 
Fibrils, collagen, 114 

fibroglia, 114 
Fibrin, 170 

stains for, 1 70 
Fibrino-serous exudations, examina- 
tion, 455 
Fibroglia fibrils, staining, 114 
Filaria Bancrofti, 406 

sanguinis hominis, 406 
Fistula, pancreatic, fluids of, 462 
Fixing cover-glass preparations, 234 
reagents, 38 



INDEX. 



539 



Fixing reagents, alcohol as, 39 
and formaldehyde as, 43 
Bethe's, for methylene-blue, 131 
boiling as, 49 
choice, 38 
chrome salts, 46 
corrosive sublimate as, 43 
Flemming's solution as, 48 
for central nervous system, 1 20 
for iron-containing pigments, 185 
formaldehyde as, 41, 247 
and alcohol as, 43 
for central nervous system, 1 20 
for frozen sections, 42 
Giemsa's solution, 44 
Helly's fluid, 38, 46 
Hermann's solution as, 48 
Kallius', for Golgi's stains, 128 
Marchi's fluid as, 48 
Muller's fluid as, 46 
Orth's fluid as, 47 
osmic acid, 48 
Pianese's solution as, 49 
sodium urate crystals as, 39 
Tellyesniczky's mixture as, 47 
Zenker's fluid as, 45, 247 

Helly's modification, 38, 46 
special organs, 147 

Flagella, staining, 239 

Claudius' method, 243 
Loffler's method, 240 
Williams' method, 241 
Zettnow's method, 244 

Flasks, Ehrlenmeyer, 206 

Flemming's solution as fixative, 48 

Flexner's method for lepros}' bacillus, 

354 
Fluids, collection of, 211 
examination of, 35 

cerebrospinal, 447 
indifferent, 35 
macerating, 35 

obtained by puncture, examina- 
tion, 455 
Fontana's stain for treponema palli- 
dum, 399 
Food for animals, 233 
Forceps, cover-glass, 234 

for autopsy, 476 
Formaldehyde and alcohol as fixa- 
tive, 43 
as fixative, 41, 247 
for central nervous system, 121 
for frozen sections, 42 
Formic-acid method, Lowit's, 102 

Ranvier's, 102 
Free hydrochloric acid, tests for, 469 
Freezing, carbon dioxid for, 20 
microtome, 20 
knife for, 20 



Freezing, Mixter's method, 20 
Fresh tissue, acetic acid treatment, 

examination of, ^ 
hydrochloric acid for, 34 
indifferent fluids for, 35 
macerating fluids for, 35 
osmic acid for, 34 
Scharlach R. for, 34 
sections of, 33 
staining, 34 
Freud's gold stain for nerve-fibers, 

130 
Friedlander's bacillus pneumoniae, 332 
Frozen sections, 52 

consistence of tissue, 53 
cutting, 52 

embedding, Wright's method, 54 
Goodpasture's method of stain- 
ing, 54 
myeLin-sheath stain for, 141 
Fuchsin, 75 
acid, 79 

and hematoxylin, Pianese's, 8$ 
and picro-nigrosin, Pianese's, 82 
malachite-green, and Martius 

yellow, Pianese's, 82 
1 and nigrosin, Pianese's, 81 
aniline-, 76 

carbol-, Ziehl-Neelson's, 75 
picro-, van Gieson's, 79 
Fungi and pathogenic bacteria, 253 
skin, examination for, 160 



Gabbet's methylene-blue, 74 
Gabbet-Ziehl-Neelson method for 

tubercle bacillus, 352 
Gall-bladder, dropsy of, fluid of, 462 
Ganglion-cells, stains for, 125 
Gangrenous exudations, examination, 

455, 456 
Gas-production of bacillus aerogenes 

capsulatus, 335 
Gastric contents, blood in, 469 
chemical examination, 469 
examination, 469 
hemin test, 469 
hydrochloric acid in, 469 
microscopic examination, 469 
tissue shreds in, 469 
Gastro-intestinal tract, 154 

inspection of, 496 
Gelatin, carmine-, as injection, 37 
dextrose, preparation, 198 
glucose, preparation, 198 
plain, preparation, 197 
Gentian-violet, 76 
carbol-, 77 
for glycogen, Lubarsch's, 178 



54Q 



INDEX. 



Gentian-violet, Stirling's, 77 
Gerlach's gold stain for nerve-fibers, 

130 
Ghoreyeb's stain for treponema palli- 
dum, 400 
Giant-cells of bone-marrow, Wright's 

stain for, 150 
Giemsa's corrosive sublimate-alco- 
hol fixative, 44 
stain for malarial parasites, 391 
for protozoa and bacteria in sec- 
tions, 392 
Wolbach's modification, 

393 
for spirochete pallida, 397 
Gillette safety razor blades for par- 
affin sections, 28 
Glanders bacillus, 314 
diagnosis, 317 

Loffler's methylene-blue for, 318 
Noniewicz's method for, 319 
Schutz's method for, 318 
staining in sections, 318 
tubercles, 316 
Globulin in cerebrospinal fluid, No- 
guchi's method, 449 
Ross- Jones method, 449 
Glucose-agar-agar, preparation,' 196 
bouillon, preparation, 192 
gelatin, preparation, 198 
Glycerin agar-agar, preparation, 196 

jelly, Kaiser's, 168 
Glycerin-albumin mixture, Mayer's, 

62 
Glycerin-alum-hematein solution, 

Mayer's, 70 
Glycogen, 177 

Best's carmine for, 178 
Langhans' iodine for, 178 
Lubarsch's gentian- violet for, 178 

iodin hematoxylin for, 178 
stains for, 177-179 
Gold as stain, 101 

for nerve-fibers, 130 
test, Lange's, of cerebrospinal fluid, 
for syphilis of central nervous 
system, 451 
Golgi's corrosive sublimate method, 
Cox's modification, 129 
stains for nervous tissues, 125-128 
Cajal's modification, 128 
fixing of, 128 
Kallius' fixative, 128 
Gonococcus, 272 
blood cultures, 276 
culture-media for, 273 
diagnosis, 274 
Gram's method for, 274 
Pappenheim's method, 277 
staining, 274, 276, 277 



Gonococcus, staphylococcus pyog- 
enes and, differentiation, 274 
streptococcus and, differentiation, 
274 
Gonorrhea, bacterium of, 272 
Gonorrheal infections, complement- 
fixation test in, 445 
Goodpasture's acid polychrome meth- 
ylene-blue solution, 75, 156 
method of staining frozen sections, 
54 
G raham's alphanaphthol - pyronin 

stain for oxydase granules, 423 
Gram's method, 249 

bacteria decolorized by, 237 
not stained by, 248 
stained by, 237, 249 
for bacteria in smear prepara- 
tions, 236 
from cultures, 234 
for gonococci, 274 
Gram-Weigert method for bacteria, 

250 
Granulation tissue, 148 
Granules in cytoplasm of plasma-cells 
and lymphocytes, Schridde's 
stain for, no 
oxydase, Graham's alphanaph- 
thol-pyronin stain for, 423 
Granuloma, coccidioidal, 376 
Green pus, bacillus of, 321 
Grippe, bacillus of, 282 
Guinea-pigs, care of, 233 
food for, 233 
inoculation of, 229 
intraperitoneal, 229 
subcutaneous, 229 
with comma bacillus, 358 
with tubercle bacilli, 351 
Giinzburg's test for hydrochloric acid, 
470 



Hair, examination of, 161 
protection of, at autopsy, 506 

Hammer, soft-iron, 476 
steel, 476 

Hand lotion in postmortem exami- 
nations, 530 

Hanging-drop, 227 
preparation, 227 
spore-formation and, 228 

Harke's method for inspection of 
nasopharynx, 519 

Harris' hematoxylin, 69, 88 

Hart's modification of Weigert's elas- 
tic tissue stain, 117 

Hatchet-chisel, 476 

Head-holder, 476 ' 

Heart, inspection of, external, 487 



INDEX. 



541 



Heart, opening of, 488 

after sudden death, 490 
removal of, 488, 491 
size of, 491 
weight of, 491 
Heidenhain's hematoxylin stains, 
method, 89 
iron hematoxylin, method, 90 
Heller's stain for myelin-sheath, 140 
Robertson's modification, 141 
Helly's modification of Zenker's fluid 

as fixing reagent, 38, 46 
Hemalum, Mayer's, 69 

method, 89 
Hematein solution, glycerin-alum-, 
Mayer's, 70 
stains, 66 
Hematogenous pigments, 183 
Hematoidin, bile-pigment-bilirubin-, 
183 
crystals in sputum, 468 
Hematoidin-bilirubin, 183 
Hematoxylin, acid, phosphomolybdic, 
70 
phosphotungstic, 71 
alcohol, Weigert's, 70 
alum, 66," 67 

method, 88 
alum-aqueous, method, 88 
and acid fuchsin, Pianese's, 83 
and light green, Pianese's, 82 
Delafield's, 68, 88 
Harris', 69, 88 
Heidenhain's, 89 
iodin-, Lubarsch's, for glycogen, 

178 
iron chlorid, Heidenhain's, 90 
Mallory's, 90 
Weigert's, 89 
phosphomolybdic-acid, for nervous 

system, 123 
phosphotungstic-acid, for nervous 
system, 123 
for neuroglia-fibers, 143 
Mallory's, 143 
stains, 66 
Hemin test, 469 
Hemocytometer, Thoma-Zeiss, 410, 

411 
Hemoglobin, 183 
Hemolysins, 428 
Hemolytic amboceptors, 428 
Hemorrhage from stomach, autopsy 

guides, 479 
Hemorrhagic exudation, examination, 

455 
Hemosiderin, 183, 184 

iron in, reaction for, 185 
Hemp twine, 476 
Hermann's solution as fixative, 48 



Hill's hanging-block method for ob- 
serving development of bacteria, 
228 

Hiss's method of staining capsules of 
bacteria for smears, 245 
serum-water medium for testing 
fermentation of carbohydrates, 
259 

Histological methods, 17 

Hobb's tea infuser, 30 

Holder, head-, 476 

Hollow slide, 227 

Honing, 28 

Hookworm disease, examination of 
feces in, 408 
eggs in feces, examination for, 470 

Hoyer's thionin for mucin, 173 

Hunt's method for bacillus diph- 
theria?, 311 

Hyalin, 174 
stains for, 174 
Unna's, 176 

Hydrocele bouillon, 276 
fluid agar, 273 

Hydrochloric acid for fresh tissue, 34 

Hydronephrosis, fluid of, 462 

Hydrophobia, 394 
Negri bodies in, 395 

Hydrothorax, albumin in, 455 

Hypodermic syringe for collecting 
fluid material, 211 



Illuminating apparatus, Abbe, 19 
Illumination for microscopic work, 

19 
Impregnations, metallic, 99. See 

also Metallic stains. 
Incisions for opening heart, 488 

to bare thorax, 483, 484 

to open abdomen, 483 
Incubator, 31 

Indian-ink method for treponema pal- 
lidum, 401 
Indifferent fluids, 35 
Inferior vena cava, opening of, 504 
Inflammatory exudations, 148 
Influenza, bacillus of, 282 
diagnosis, 285 
staining in sections, 286 
Injections, 36 

Berlin blue as, 37 

blue coloring as, 36 

carmine as, 36 

carmine-gelatin as, 37 

cold, 36 

warm, 37 
Inoculation of animals, 229 
for diagnosis, 461 
quantity of bacteria used, 232 



542 



INDEX. 



Inoculation of guinea-pigs, 229 
intraperitoneal, 229 
subcutaneous, 229 
with comma bacillus, 358 
with tubercle bacilli, 351 
of media, 216 
of mice, 231 
of rabbits, 230 
intravenous, 230 
Inoscopy, 457, 460 
Instruments for postmortem exami- 
nation, 473 
metal, for histologic work, 31 
Internal examination of body, 482 
Intestine, 154 
opening of, 498 
removal of, 497 
Intraperitoneal inoculation of guinea- 
pigs, 229 
Intravenous inoculation of rabbits, 

230 
Iodin and sulphuric acid for amyloid, 
181 
for cellulose, 181 
for cholesterin crystals, 181 
for corpora amylacea, 181 
for starch granules, 181 
as stain, 8$ 
for amyloid, 180 
Langhans' method for permanent 
mounts, 181 
Langhans', for glycogen, 178 
Iodin-green for amyloid, 182 
Iodin-hematoxylin, Lubarsch's, for 

glycogen, 178 
Iron as pigment, 184 
hematoxylin, chlorid of, Mallory's 
method, 90 
Heidenhain's method, 90 
Weigert's, 89 
in hemosiderin, reaction for, 185 
tests, 185 

for iron-containing pigments, 185 
Iron-alizarin-toludin-blue for neurog- 
lia fibrils, 146 
Iron-containing pigments, 184 
fixing reagents for, 185 
iron tests for, 185 
stains for, 184 
Isolation of bacterium in pure culture 
from mixed growth, 217 

Jaundice, opening duodenum in, 498 

removal of liver in, 499 
Jousset's method for tubercle bacillus 

in serous fluids, 456, 460 

Kaiserltng's method to preserve 
natural colors in museum prepara- 
tions, 162 



Kaiserling's method to preserve nat- 
ural colors in museum prepara- 
tions, Pick's modification, 1 63 

Kaiser's glycerin jelly, 168 

Kallius' fixing method for Golgi's 
stains, 128 

Karyomitosis, staining for, 97 

Keratohyalin, 176 

Kidney, 153 
new-growths in, urine indications,, 

471 
removal of, 500 
size, 502 
weight, 502 

Klotz's stain for fat acid crystals, 168 

Knives, amputation, 474 
autopsy, 474 
cartilage, 474 
for cutting sections, 22 
microtome, 27 

Koch's bacillus, 342. See also Bacil- 
lus tuberculosis. 
for inoculation of guinea-pigs 
with comma bacillus, 358 

Kuhne's method for tubercle bacillus, 
352 
methylene-blue, 74 

Kulschitzky-Wolter's modification of 
Weigert's myelin-sheath stain, 139 



Laboratory outfit, 17 

Lactose-litmus agar-agar preparation, 
196 

Lange's colloidal gold test of cerebro- 
spinal fluid for syphilis of central 
nervous system, 451 

Langhans' iodin for glycogen, 178 
method for permanent mounts of 
amyloid with iodin, 181 

Lavender oil as clearing reagent, 104 

Leishman's stain for blood, 385 

Lenhossek's stain for tigroid bodies, 

125 
Lens, oil-immersion, 19 
Leprosy, bacillus of, 353 

Baumgarten's stain for, 354 
cultures, 353^ 
Flexner's stain for, 354 
occurrence, 354 
pathogenesis, 354 
staining in sections, 354 
tubercle bacillus and, differentia- 
tion, 348 
Leptothrix of Parinaud's conjuncti- 
vitis, Verhoeff's modified Gram 
stain for, 250 
Leucocytes and myelocytes, 
Schridde's stain for, 150 
counting of, 410, 413 



INDEX. 



543 



Leucocytes in blood, 410 

in cerebrospinal fluid, 488 
Levaditi's stain for treponema palli- 
dum, 401 
Liborious' method for anaerobes, 221 
Light green and hematoxylin, Pi- 

anese's, 82 
Lithium carmine method, 92 

Orth's, 72 
Litmus-milk preparation, 201 
Liver, 154 

removal of, 499 
size of, 500 
weight of, 500 
Lobar pneumonia, bacterium of, 

263 
Loffler's bacillus, 304 
staining, 309 

Hunt's method, 311 
toxin-production, 307 
method for staining flagella, 240 
methyl ene-blue, 74 
, for bacteria, 248 

for glanders bacillus, 318 
mixture, preparation, 198 
Loop, platinum, 213 
Lotion, hand, in postmortem exami- 
nations, 530 
Lowit's formic-acid method, 102 
Lubarsch's gentian-violet for glyco- 
gen, 178 
iodin-hematoxylin f or glycogen, 178 
Luer's double rachiotome, 476 
Lugol's solution, 83 
Lumbar puncture, 447 
Lung, 149 

incisions into, 493 
inspection of, 487 
removal of, 492 
Lymphocytes after Wright's stain, 
420 
cytoplasm of, Schridde's method 
for demonstrating granules in, 
no 



Macerating fluids, 35 
Malachite-green, acid fuchsin, and 

nigrosin, Pianese's, 81 
Malarial organisms, 385 

ameboid movements, 385, 386 

changes in, 386 

cycle, 387 

development, 385 

double infections, 389 

estivo-autumnal, 385 
cycle, 388 

Giemsa's stain for, 391 

in blood, examination, 389 

pigment in, 386 



Malarial organisms, quartan, 385 
cycle, 387 
Romanowsky's method for, 389, 

390, 391, 418 
segmentation of, 386 
tertian, 385 
cycle, 387 
varieties, 385 
Wright's stain for, 389 
Malignant edema, bacillus of, 340 
Mallinckrodt's pyroxylin, 56 
Mallory's acid-fuchsin stain for fibro- 
glia fibrils, 115 
aniline-blue for connective tissue, 

in 
chlorid of iron hematoxylin 

method, 90 
eosin and methylene-blue stain, 94 
instantaneous alum-hematoxylin, 

68,88 
phosphomolybdic acid hematoxy- 
lin, 70 
phosphotungstic acid hematoxylin, 

7i, 143 
stain for actinomyces bo vis, 371 
for diphtheria bacillus, 311 
for fibroglia fibrils, 115 
for neuroglia fibers, 143 
Mall's stain for reticulum, 113 
Mannite litmus-agar, 301 
Marchi's fluid as fixative, 48 

stain for fatty degeneration of ner- 
vous tissue, 147 
Marrow-bone, 149 

inspection of, 505 
Martius yellow, malachite-green, and 

acid fuchsin, Pianese's, 82 
Mast-cells, 108 

after Wright's stain, 421 
stains for, 108 
Mayer's alcoholic carmine, 72 
glycerin-albumin mixture, 62 
glycerin-alum-hematein solution, 

70 
hemalum, 69 
acid, 70 
method, 89 
muchematein, 172 
stain for amyloid, 182 
Mcjunkin's tube for collecting blood 

for cultures, 425 
Mechanical stage for microscope, 19 
Megakaryocytes, Wright's stain for, 

150 
Melanin, 183 
Meningitis, bacterium of, 279 

diagnosis, 282 
Mesenteric veins, injection of bac- 
teria into, 230 
Mesentery, removal of, 497 



544 



INDEX. 



Metal instruments for histologic 

work, 31 
Metallic stains, 99 

gold as, 101 

osmic acid as, 102 

osmium tetroxid as, 102 

perosmic acid as, 102 

silver as, 99 
Methemoglobin, 183 
Methylene-blue, 74 

acid polychrome, Goodpasture's, 

75, 156 
alkaline, Unna's, 74 
and eosin in borax solution, Pian- 
ese's, 80 
method, 94 
borax, Sahli's, 75 
for nerve-fibers, 131 
Kiihne's, 74 
Loffler's, 74 

for bacteria, 248 
for glanders bacillus, 318 
polychrome, Unna's, 74 

for mucin, 173 
Sahli's borax, 75 
Methyl-green and pyronin stain, 
Pappenheim's, for bacteria 
in smear preparations, 236 
Unna-Pappenheim, for bac- 
teria, 249 
for plasma-cells, no 
Methyl-violet, 76 
for amyloid, 181 
shellac, 529 
Mice, care of, 233 
food for, 233 
inoculation of, 231 
Micrococcus catarrhalis, 277 
lanceolatus, 263 
diagnosis, 267 
of sputum septicemia, 263, 266 

diagnosis, 267 
pneumoniae crouposae, 263 
tetragenus, 278 
Microscopes, 18 
Microscopic examination of bacteria 

in smear preparations, 235 
Microtome celloidin, 22 
drop-bottle on, 24 
freezing, 20 

knife for, 22 
knives, 27 
Minot's precision, 24 

wheel, 24 
paraffin, 24 
Microtomes, 20 
Middle ear, inspection of, 518 
Milk, litmus-, preparation, 201 
Minot precision microtome, 24 
wheel microtome, 24 



Mitochondria, 107 

Bensley's stain for, 107 

staining, 107 
Mitosis, 97 

Mixter's freezing method, 20 
Moeller's method for staining spores, 

238 
Mononuclear leucocytes after 

Wright's stain, 421 
Morris' stain for skin parasites, 161 
Motility of bacteria, determination, 

227 
Mounting reagents, 105 
Mouse-holder, 232 
Muchematein, Mayer's, 172 
Mucin, 171 

aniline dyes for, 172 

Hoyer's thionin for, 173 

pseudo-, 173 

stains for, 172 

test for, 171 

Unna's polychrome methylene-blue 
for, 173 
Muir's method of staining capsules of 

bacteria for smears, 246 
Muller's fluid as fixative, 46 
Muscle-cells, smooth, staining, ir9 

striated, staining, 119 
Museum preparations, 162 

natural colors of, preserving, 162, 
163, 164 
Musgrave and Clegg's method of cul- 
tivating amoebae, 384 
Myelin-sheath, Exner's stain for, 140 

fatty degeneration of, stains for, 

147 
Heller's stain for, 140 

Robertson's modification, 141 
stains for, 135 

for frozen sections, 141 
Weigert's stain for, 135 

Kulschitzky-Wolter's modifi- 
cation, 139 
Pal's modification, 138 
Myelocytes after Wright's stain, 421 
and leucocytes, Schridde's stain 
for, 150 
Myelotome, 474 
Myoglia fibrils, Benda's stain for, 



Nasopharynx, inspection of, 519 
Neck, block for, at autopsy, 476 

organs of, removal of, 494 
Necrosis, 169 
Needles, autopsy-, 476 
Neelson-Gabbet-Ziehl method for 

tubercle bacillus, 352 
Negri bodies, 395 



INDEX. 



545 



Negri staining, 395 

Neisser's method for bacillus diph- 

theriae, 310 
Nerve-fibers, aniline-blue for, 124, 

axis-cylinders of, gold stain for, 

101 
chlorid of iron and dinitroresorcin 

for, 134 
fatty degeneration of, stains for, 

147 
gold stain for, 130 
methylene-blue for, 131 
myelin-sheath of, stains for, 135 
Nervous system, central. See Cen- 
tral nervous system. 
Neuroglia fibers, Mallory's stain for, 

phosphotungstic acid hematox- 
ylin for, 143 
stains for, 142 
Weigert's stain for, 144 
fibrils, Benda's stain for, 146 
iron-alizarin-toluidin for, 146 
Neutral balsam, 105 

carmine, 72 
Neutrophils, polynuclear, after 

Wright's stain, 420 
New practical staining-dish, 30 
Newborn, examination of, 520 

weight of organs of, 522 
Nigrosin for nervous tissue, 124 
malachite-green, and acid fuchsin, 
Pianese's, 81 
Nissl bodies, stains for, 1 24 
Nitrate of silver as stain, 101 
Nitric acid and phloroglucin in decal- 
cification, 51 
in decalcification, 50 
Noguchi method for globulin in cere- 
brospinal fluid, 449 
reaction in syphilis, 427 
amboceptor, 428, 433 
antihuman, 433 
antisheep, 435 
standardization of, 437 
antigen, 429, 435 

standardization of, 439 
apparatus needed, 432 
centrifuge in, 433 
complement, 427, 433 
corpuscle suspension, 437 
interpretation of results, 441 
patient's serum, 436 
preparation of reagents, 432 
saline solution in, 433 
standardization of reagents, 

437 
technique, 440 
water-bath in, 433 

35 



Noniewicz's method for glanders 

bacillus, 319 
Nuclear stains, 87 

alum-hematoxylin, aqueous, 88 

instantaneous, 88 
aniline dyes as, 93 
carmine, 92 

combination, method, 96 
Delafield's alum-hematoxylon, 88 
Ehrlich's acid hematoxylin, 82 
Harris's alum-hematoxylin, 88 
Heidenhain's hematoxylin, 89 
hematoxylin, 88, 89, 90 
Mallory's chlorid of iron hema- 
toxylin, 90 
. Mayer's hemalum, 89 
Nutrition, general, in autopsy, 481 



Oidiomycosis, 376 
Oil, aniline, as clearing reagent, 104 
of bergamot as clearing reagent, 104 
of cedar as mounting reagent, 106 

in paraffin embedding, 59 
of cedar-wood as clearing reagent, 

104 
of cloves and thyme as clearing 
reagent, 104 
as clearing reagent, 104 
of lavender as clearing reagent, 104 
of thyme as clearing reagent, 104 
and cloves as clearing reagent, 
105 
Oil-immersion lens, 19 
Olein, 165 
Oleum origani cretici as clearing 

reagent, 104 
Orbit, examination of contents, 517 
Orcein, 83 
Orth's fluid as fixative, 47 

lithium carmine, 72 
Osmic acid as fixative, 48 
as stain, 102 
for fresh tissue, 34 
Osmium for fat, 166 

tetroxid as stain, 102 
Ovarian cysts, fluid of, 461 
Ovaries, opening of, 503 

weight of, 503 
Ox-bile method for cultivating ty- 
phoid bacilli, 298 
Oxydase granules, Graham's alpha- 
naphthol-pyronin stain for, 423 
reaction, Schultze's, 422 
Oxygen, cultivation without, 220 



Paxmitin, 165 

Pal's modification of Weigert's mye- 
lin sheath stain, 138 



546 



INDEX. 



Pancreas, 155 

cross-sections of, 499 
removal of, 497 
stains for, 155 
Pancreatic cysts, fluid of, 462 
Pappenheim's method for staining 
gonococcus, 277 
methyl-green-pyronin stain for 

mast-cells, no 
pyronin and methyl-green mixture 
for bacteria in smear prepara- 
tions, 236 
Paracarmine, 72 
Paraffin bath, 24 

embedding, 55, 59, 66 

method for serial sections, 61, 

66 
microtome, 24 

sections, attaching to slides, 62 
cutting, 61, 62 

Gillette safety razor blades for, 
28 
Parasites, animal, 381 
examination, 385 
in sputum, 466 
estivo-autumnal, 385 

cycle, 388 
quartan, 385 
cycle, 387 
. tertian, 385 
cycle, 387 
vegetable, in sputum, 466 
Paratyphoid bacilli, 299 

examination of feces for, 302 
Parhemoglobin, 183 
Parinaud's conjunctivitis, leptothrix 
of, Verhoeff 's modified Gram stain 
for, 250 
Parovarian cysts, fluid in, 461 
Pathogenic bacteria and fungi, 253 
Pathological fluids, collection of, 210, 
211 
material, collection of, for ex- 
amination, 209 
products, 164 
Pathology, clinical, 410 
Pelvic organs, removal of, 502 
Penis, removal of, 504 
Pepton solution, Dunham's prepara- 
tion, 202 
Pericardium, opening of, 487 
Peritonitis, acute, autopsy guides, 

479 
Perosmic acid as stain, 102 
Pertussis, bacillus of, 286 
Petri dishes, 219 

oblong rectangular, 30 
for discrete colonies, 218 
plate method for isolation of bac- 
terium, 218 



Petrifaction, stains for, 187 
Petroff's method for cultivation of 
tubercle bacilli from sputum and 
feces, 345 
Petrous bone, removal of, 518 
Pfeiffer's method for inoculation of 
guinea-pigs with comma bacillus, 
359 
reaction of comma bacillus, 359 
Phenolphthalein as indicator in ad- 
justment of reaction, 203 
Phloroglucin and nitric acid in decal- 
cification, 51 
Phloroglucin-vanillin test for hydro- 
chloric acid, 470 
Phosphomolybdic acid hematoxylin, 
70 
for nervous system, 123 
Phosphotungstic acid hematoxylin, 

7i 
for nervous system, 123 
for neuroglia fibers, 143 
Mallory's, 143 
Pia, removal of, 491 

stripping off of, 510 
Pianese's acid fuchsin and hematoxy- 
lin, 83 
and picro-nigrosin, 82 
carmine and picro-nigrosin, 80 
hematoxylin and light green, 82 
malachite-green, acid fuchsin, and 
Martius yellow, 82 
and nigrosin, 81 
methylene-blue and eosin in borax 

solution, 80 
solution as fixative, 49 
stains, 80 
Pick's modification of Kaiserling's 
method to preserve natural colors 
in museum preparations, 163 
Picric acid as stain, 78 

method, 96 
Picro-fuchsin, van Gieson's, 79 
Picro-nigrosin, 79 

and acid fuchsin, Pianese's, 82 
and carmine, Pianese's, 80 
Pigments, 183 

autochthonous, 183 
extraneous, 184 
hematogenous, 183 
iron-containing, 184 
fixing reagents for, 185 
iron tests for, 185 
stains for, 184 
Pitre's section of brain, 514 
Plague, bubonic, bacillus of, 323 

diagnosis, 327 
Plain agar-agar, precipitates in, 195 
preparation, 193 
gelatin, preparation, 197 



INDEX. 



547 



Plasma-cells, cytoplasm of, Schridde's 
method for demonstrating 
granules in, no 
staining, no 
Plasmodium malariae, 385. See also 

Malaria organisms. 
Plate method, Petri's, for discrete 

colony, 218 
Plate-cultures, anaerobic, 223 
Zinsser's, for anaerobes, 224 
Platinum wire, 213 
Pleural adhesions, 487, 492 

cavities, inspection of, 486 
Pneumococcus, 263 
capsule of, 265 
staining, 237 
diagnosis, 266 
streptococcus and, differentiation, 

258 
types of, determination, 268 
Pneumonia, bacterium of, 263 

diagnosis, 267 
Polychrome methylene-blue, Unna's, 

74 
Postmortem discolorations, 481 

examinations, 472. See also Au- 
topsy. 

rigidity, 482 
Potash, caustic, as macerating fluid, 

35 

Potato-cultures, Bolton's prepara- 
tion, 201 

Precipitates in agar-agar, 195 

Primary anterior incision, 483 

Private autopsy, 479 

Probes, 476 

Proteids in cerebrospinal fluid, in- 
crease tests, 449 

Proteus group, 304 
mirabilis, 304 
vulgaris, 304 
Zenkeri, 304 

Protoplasm of cells, gold stain for, 
101 

Protozoa in sections, Giemsa's stain, 
392 

Prussian (Berlin) blue, 186 

Pseudo-mucin, 173 

Puncture fluids, examination, 455 
lumbar, 447 

Pure culture, isolation of bacterium 
in, from mixed growth, 217 
obtaining of, 216 

Purulent exudation's, examination, 
455, 456 

Pus, green, bacillus of, 321 
tubercle bacilli in, 350 

Putrefaction, bacteria of, 304 

Putrid exudations, examination, 455, 
456 



Pyronin and methyl-green mixture, 
Pappenheim's, for bacteria in 
smear preparations, 236 

Pyroxylin, Mallinckrodt's, 56 



Quartan parasite, 385 
cycle, 387 



Rabbits, anesthetizing, 230 
care of, 233 
food for, 233 
inoculation of, 230 
intravenous, 230 
Rabies, 394 

Negri bodies in, 395 
Rachiotome, double, Luer's, 476 
Ranvier's formic-acid method, 102 

macerating fluid, 35 
Ravaut's method for examination of 

serous fluids, 456, 457 
Ray-fungus. See also Actinomyces 

bovis. 
Reaction, complement-fixation, in 
echinococcus infections, 446 
in gonorrheal infections, 445 
Noguchi, in syphilis, 427. See also 

Noguchi reaction. 
of culture-media, titration in ad- 
justment, 202 
Schultze's oxydase, 422 
Wassermann, in syphilis, 427. See 
also Wassermann reaction. 
Rectum, opening of, 503 

removal of, 503 
Red blood-corpuscles, after Wright's 
stain, 420 
counting of, 410 
in blood, 410 
blood-globules in sputum, 465 
Relapsing fever, spirochetes of, 396 
Renal cysts, fluid of, 462 
Resorcin test, Boas', for hydrochloric 

acid, 470 
Respiration, determination of act oc- 
curring, 521 
Reticulum, staining, in, 113 
Rhinoscleroma, bacillus of, staining, 

334 
Ribbons of sections, 62, 65 
Rigidity, postmortem, 482 
Ringworms, medium for, 162 
Ripening, 66 
Robertson's modification of Heller's 

myelin-sheath stain, 141 
Roehl's method for calcium deposits, 

187 
Romanowsky's method for malarial 

organisms, 389, 390, 391, 418 



548 



INDEX. 



Ross-Jones method for globulin in 
cerebrospinal fluid, 449 

Round worms, 406 

Rubber gloves for autopsy, 478 

Running water for washing speci- 
mens, 28 

Russell medium in examination of 
feces for typhoid, paratyphoid, and. 
dysentery bacilli, 303 

Saathoff's stain for bacteria, 249 

Sabouraud's medium for ringworms, 
162 

Safranin, 77 

aniline, Babes', 78 
as nuclear stain, method, 93 
for karyomitotic staining, 99 
Schaffer's, for differentiating bone 
from cartilage, 158 

Sahli's borax methylene-blue, 75 

Saline solution in serum diagnosis of 
syphilis, 433 

Saw, bone, 474 

Scales for autopsy, 474 

Scalpels, 474 

Scarlet fever, 312 

Gram-Weigert stain for, 313 

Schaffer's safranin for differentiating 
bone from cartilage, 158 

Schallibaum's solution, 62 

Scharlach R. for fat, 165, 167 
for fresh tissue, 34 

Schering's celloidin, 56 

Schistosomum haematobium, 405 

Schmorl's method of differentiating 
bone from cartilage, 159 

Schridde's method for demonstrating 
granules in cytoplasm of plas- 
ma-cells and lymphocytes, no 
of removing precipitate from 
formaldehyde, 42 
stain for granulations of myelo- 
cytes and leucocytes, 150 
solution for decalcification, 51 

Schultze's oxydase reaction, 422 

Schiitz's method for glanders bacil- 
lus, 318 

Scissors, 474 

Sedimentation for tubercle bacillus in 
sputum, 349 

Segmentation of Plasmodium mala- 
rias, 386 

Semilunar ganglia, location, 504 

Septicemia, sputum-, micrococcus of, 
263, 266 

Serial sections, 63 

by celloidin method, 63 
by paraffin method, 66 

Serofibrinous exudations, examina- 
tion, 455 



Serous exudations, examination, 455 
fluids, examination, 456 

tubercle bacillus in, inoscopy for, 
460 
Serum, artificial, 35 
blood-, collection of, 198 
cultures on, 212 

preparation, 212 
preparation, 198 
reaction in typhoid, 297, 300 
diagnosis of syphilis, 427 
reaction of bacillus typhosus, 297, 
300 
Serum-water medium, Hiss's, for 
testing fermentation of carbohy- 
drates, 259 
Shiga bacillus, 300 
Silver as pigment, 184 

as stain, 99 
Simple anaerobic plate-cultures, 223 
staining of bacteria in smear prep- 
arations, 235 
Skeleton, development of, 481 
Skin, condition of, in autopsy, 481 
parasites of, staining, 161 
staining, 160 
Skull, infants, opening of, 507 

opening of, 506 
Slant culture, 194, 198, 201, 206 
Slee's restitution of calvarium, 507 
Slides, 28 
attaching celloidin sections to, 58 

paraffin sections to, 62 
cleaning, 530 
hollow, 227 
old, cleaning, 530 
storage of, 31 
Smear preparations, bacteria in, 
staining, 234 
of blood on slides, 417 
of Negri bodies, 395 
of treponema pallidum, 397 
Smegma bacillus, tubercle bacillus 

and, differentiation, 348 
Smith and Brown's method of culti- 
vating tubercle bacilli from spu- 
tum, 346 
Smith's method of staining bacteria 
in sputum, 237 
capsules of bacteria for smears, 
244 
Smooth muscle-cells, staining, 119 
Sodium urate crystals as fixative, 39 
Spatulas, 31 
Special organs, examining, 147 

stains, 107 
Specific gravity of puncture fluids, 

455 
Spinal column, removal of, 505 
cord, removal of, 515 



INDEX. 



549 



Spirals, Curschmann's, in sputum, 

464 
Spirillum of Asiatic cholera, 355. See 

also Comma bacillus. 
Spirochete pallida, Benian's method, 

399 

Burri's India-ink method, 401 

Eyenes and Sternberg's stain, 
402 

Fontana's method, 399 

Ghoreyeb's stain, 400 

Giemsa's stain, 397 

in smear preparations, 397 

Levaditi's stain, 401 

Wright's stain, 397 
Spirochetes of relapsing fever, 396 
Spleen, 149 

removal of, 496 
sections, 149 
smear preparations, 153 
staining, 149 
Spores, formation of, hanging drop in 

study, 228 
in media, destroying, 206 
staining, 238 

Abbott's method, 238 

Moeller's method, 238 
vitality of, 206 
Sporotrichosis, 373 
Sporotrichum Schenckii, 373 

diagnosis, 376 
Sputum, animal parasites in, 466 
bacteria in, Smith's stain for, 237 
cultivation of tubercle bacilli from, 

Petroff's method, 345 
blood-globules in, 465 
caseous masses in, 464 
cells in, 465 

Charcot-Leyden crystals in, 467 
cholesterin crystals in, 468 
concretions in, 465 
crystals in, 467, 468 
Curschmann's spirals in, 464 
Dittrich's plugs in, 464 
elastic fibers in, 465 
epithelial cells in, 465 
examination of, 463 
fatty detritus in, 465 
fatty-acid crystals in, 469 
fibrinous casts of bronchioles in, 

464 
hematoidin crystals in, 468 
influenza bacilli in, 285 
macroscopic appearance, 464 
microscopic examination, 465 
parasites in, 466 
tissue shreds in, 465 
tubercle bacilli in, 466 

antiformin method of culti- 
vating, 346, 349 



Sputum, tubercle bacilli in, examina- 
tion for, 348 
vegetables parasites in, 466 
Sputum-septicemia, diagnosis, 267 

micrococcus of, 263, 266 
Stab culture, 196, 205 

deep, 223 
Staining, 84. See also Stains. 
differentiation by, 86 
dishes, 29 
concave, 30 
crystallizing, 30 
Hobb's tea infuser, 30 
new practical, 30 
Petri's oblong rectangular, 30 
in mass, 97 
method, 84 
steps in, 87 
Stains, 66 
acetic acid, for fat, 166 
acid hematoxylin, phosphomo- 
lybdic, 70 
phosphotungstic, 71 
alcoholic carmine, Mayer's, 72 
hematoxylin, Weigert's, 70 
Algeri's, for fatty degeneration of 

nervous tissue, 147 
alphanaphthol-pyronin, Graham's, 

for oxydase granules, 423 
alum carmine, 71 
method, 92 
cochineal, 72 
method, 92 
alum-hematoxylin, 66, 67 

aqueous, method, 88 
aniline, 73 

blue, for nervous system, 1 24 

Stroebe's, 134 
for bacteria in sections of tissue, 

246 
for mucin, 172 
safranin, Babes', 78 
aniline-fuchsin, 76 
aniline-gentian-violet, Ehrlich's, 

77 
Babes' aniline safranin, 78 
Baumgarten's, for leprosy bacillus, 

354 
Benda's, for fat acid crystals, 168 

for myoglia fibrils, 120 

for neuroglia fibrils, 146 
Benian's, for treponema pallidum, 

399 

Bensley's, for mitochondria, 107 
Best's carmine, for glycogen, 178 
Bielschowsky's, for axis-cylinders 
and neurofibrils, 132 

for collagen fibrils, 113 
Biondi-Heidenhain's, 79 

method, 96, 97 



55o 



INDEX. 



Stains, Bismarck-brown, 78 

for amyloid, 182 
Burri's, for treponema pallidum, 

401 
Cajai's double method for nervous 

tissue, 128 
carbol-fuchsin, Ziehl-Neelson's, 75 
carbol-gentian-violet, 77 
carmine, 71, 92 

alcoholic, Mayer's, 72 

alum, 71 
method, 92 

Best's, for glycogen, 178 

lithium, Orth's, 72 

method, 92 

neutral, 72 
chlorid of gold as, 101 
for nerve-fibers, 130 

of iron and dinitroresorcin for 
nerve-fibers, 134 
Claudius', for flagella, 243 
cochineal alum, 72 

method, 92 
combination, 79 

Cox's modification of Golgi's corro- 
sive sublimate method, 129 
Delafield's hematoxylin, 68 

method, 88 
differential, 86 
diffuse, 78 
dinitroresorcin and chlorid of iron 

for nerve-fibers, 134 
. Ehrlich's aniline-gentian-violet, 77 

for mast-cells, 108 

for tubercle bacillus, 352 

triple, for blood, 417 
Ehrlich-Westphal, for mast-cells, 

109 
eosin, 78 

and methylene-blue, Mallory's, 
94 

method, 95 
Exner's, for myelin-sheath, 140 
Eyenes and Sternberg's, for tre- 
ponema pallidum, 402 
Flexner's, for leprosy bacillus, 354 
Fontana's, for treponema pallidum, 

399 . 
for actinomyces bovis, 371 
for albuminous degenerations, 164 
for amyloid, 180 

for axis-cylinder processes, 125,129 
for bacillus diphtherise, 309 

typhosus, 296, 297 
for bacteria in sections, 246, 392 
in smear preparations, 234 
from cultures, 234, 235 
Gram's, 236 
bacteria decolorized by, 
237 



Stains for bacteria in smear prep- 
arations, Gram's, bacteria 
stained by, 237 
microscopic examination, 

235 
Pappenheim's pyronin and 
methyl-green mixture, 236 
simple, 235 

for blood, 417 

for blood-films, 420 

for bone, 157, 158, 159 

for bone-marrow, 150 

for calcification, 187 

for calcium deposits, 187 

for capsules in sections, 251 
Smith's method, 251 

for cartilage, 157 

for caseation, 170 

for cellulose, 181 

for central nervous system, 120. 
See also Central nervous system. 

for chancroid. bacillus, 318, 319 

for cholesterin crystals, 169, 181 

for cloudy swelling, 164 

for collagen fibrils, 113 
reticulum, 113 

for colloid, 1 74 

for connective tissue, 114 

for corpora amylacea, 181 

for degenerations of nervous sys- 
tem, 146 

for dendritic processes, 125 

for elastic fibers, .116 

for entamoeba, 382 

for fat, 165 

for fat-acid crystals, 168 

for fatty degeneration of myelin- 
sheath, 147 
of nerve-fibers, 147 

for fibrin, 170 

for fibroglia fibrils, 114 

for flagella, 239. See also Flagella, 
staining of. 

for fresh tissue, 34 

for ganglion-cells, 125 

for glanders bacillus, 318 

for glycogen, 177-179 

for gonococci, 274, 276, 277 

for granulations of myelocytes and 
leucocytes, 150 

for hyalin, 174 

for influenza bacillus, 286 

for iron in hemosiderin, 184 

for iron-containing pigments, 184 

for karyomitosis, 97 

for leprosy bacillus, 354 

for Loffler's bacillus, 309, 310 

for malarial parasites, 385 

for mast-cells, 108 

for mitochondria, 107 



INDEX. 



551 



Stains for mitosis, 97 
for mucin, 171 
for myelin-sheath, 135 

for frozen sections, 141 
for necrosis, 169 
for Negri bodies, 395 
for nerve-fibers, 129 
for neuroglia-fibers, 142 
for Nissl bodies, 124 
for oxydase granules, 423 
for pancreas, 155 
for petrifaction, 187 
for plasma-cells, 109 
for pneumococcus capsule, 237 
for protozoa in sections, 392 
for reticulum, n, 113 
for rhinoscleroma bacillus, 334 
for skin, 160 

parasites, 161 
for smooth muscle-cells, 119 
for spirochaeta pallida, 397 
for spleen, 149 
for spores, 238 

Abbott's, 238 

Moeller's, 238 
for starch-granules, 181 
for striated muscle-cells, 119 
for syphilis bacillus, 397 
for terminal processes, 129 
for tigroid bodies, 124 
for treponema pallidum, 397 
for tubercle bacillus, 351 

in celloidin sections, 353 
formic-acid, 102 

Freund's gold, for nerve-fibers, 130 
fuchsin, 75 

Gabbet's methylene-blue, 74 
gentian- violet, 76 

for glycogen, Lubarsch's, 178 
Gerlach's gold, for nerve-fibers, 

130 
Ghoreyeb's, for treponema palli- 
dum, 400 
Giemsa's, for malarial parasites, 

39i 
for protozoa and bacteria in sec- 
tions, 392 
Wolbach's modification, 

393 
for treponema pallidum, 397 
glycerin-alum-hematein, Mayer's, 

70 
gold as, 101 

for nerve-fibers, 130 
Golgi's corrosive sublimate method, 
Cox's modification, 129 
for nervous tissue, 125-128 

Kallius' fixing method, 128 

Goodpasture's acid polychrome 

methylene-blue solution, 75, 156 



Stains, iGraham's alphanaphthol-py- 
ronin, for oxydase granules, 423 
Gram's, 249 

bacteria not stained by, 248 

stained by, 249 
Verhoeff's modification, 250 
Gram-Weigert, for bacteria, 250 
Hart's modification of Weigert's, 

for elastic tissue, 117 
Heidenhain's hematoxylin, 89 

iron hematoxylin, 90 
Heller's, for myelin-sheath, 140 

Robertson's modification, 141 
hemalum, Mayer's, 69 

method, 89 
hematein, 66 

glycerin-alum-, Mayer's, 70 
hematoxylin, 66 

acid, phosphomolybdic, 70 

phosphotungstic, 71 
alcohol, Weigert's, 71 
alum, 67 

alum-aqueous, method, 88 
chlorid of iron, Mallory's 

method, 90 
Delafield's, 68 
method, 88 
Heidenhain's, 89 
iodin-, Lubarsch's, for glycogen, 

178 
iron, Heidenhain's, 90 

Weigert's, 89 
phosphomolybdic acid, 70 
phosphotungstic acid, for neu- 
roglia-fibers, 143 
Weigert's alcohol, 70 
Hiss's, for capsules of bacteria for 

smears, 245 
Hoyer's thionin for mucin, 173 
iodin, 83 

and sulphuric acid, for amvloid, 
181 
for cellulose, 181 
for cholesterin crystals, 181 
for corpora amylacea, 181 
for starch granules, 181 
for amyloid, 180 
for glycogen, 177 
Langhans', for glycogen, 178 
iodin-green, for amyloid, 182 
iodin-hematoxylin, Lubarsch's, for 

glycogen, 178 
iron-alizarin-toluidin-blue, for neu- 
roglia fibrils, 146 
Kallius' fixing method for Golgi's, 

128 
Klotz's, for fat-acid crystals, 168 
Kuhne's, for tubercle bacillus, 
352 
methylene-blue, 74 



552 



INDEX. 



Stains, Kulschitzky-Wolter's modifi- 
cation of Weigert's, for myelin- 
sheath, 139 
Langhans', for permanent mounts 
of amyloid, 181 
iodin, for glycogen, 178 
Leishman's, for blood, 418 
Lenhossek's, for tigroid bodies, 

125 
Levaditi's, for treponema pallidum, 

401 
lithium carmine, 92 

Orth's, 72 
Loffler's methylene-blue, 74 
for bacteria, 248 
for glanders bacillus, 318 
Lowit's formic acid, 102 
Lubarsch's gentian--violet, for gly- 
cogen, 178 
iodin-hematoxylin, for glycogen, 
178 
Lugol's solution, 83 
Mallory's acid-fuchsin, 115 

aniline blue, for connective tis- 
sue, in 
chlorid of iron hematoxylin 

method, 90 
eosin and methylene-blue, 94 
for actinomyces bo vis, 372 
for diphtheria bacillus, 31 
for fibroglia fibers, 114 
for neuroglia fibers, 143 
hematoxylin, 68, 88 
phosphotungstic acid hematoxy- 
lin, 71 
Mall's, for reticulum, 113 
Marchi's, for fatty degeneration of 

nervous tissue, 147 
Mayer's carmine, alcoholic, 72 
for amyloid, 182 
glycerin-alum-hematein solution, 

70 
hemalum, 69 
acid, 70 
method, 89 
muchematein, 172 
metallic, 99. See also Metallic 

stains. 
methylene-blue, 74 
and eosin, method, 94 
for nerve-fibers, 131 
Loffler's, for bacteria, 248 

for glanders bacillus, 318 
Unna's polychrome, for mucin, 

i73 
methyl-green-pyronin for bacteria, 
249 
for plasma-cells, no 
methyl-violet, 76 
for amyloid, 181 



Stains, Morris', for skin parasites, 

161 
muchematein, Mayer's, 172 
Muir's, for capsules of bacteria for 

smears, 246 
nigrosin, for nervous system, 124 
Nissl's, 124 
nitrate of silver, 101 
Noniewicz's, for glanders bacillus, 

3i9 
nuclear, 88. See also Nuclear 

stains. 
orcein, 83 

Orth's lithium carmine, 72 
osmic acid, 102 
osmium, for fat, 166 

tetroxid, 102 
Pal's modification of Weigert's, for 

myelin-sheath, 138 
Pappenheim's method for gono- 

coccus, 277 
paracarmine, 72 
perosmic acid, 102 
phosphomolybdic acid hematoxy- 
lin, 70 
for nervous system, 1 23 
phosphotungstic-acid hematoxylin, 

7i 
for nervous system, 1 23 
for neuroglia-fibers, 143 
Mallory's, 143 
Pianese's, 80 
picric acid, 78 

method, 96 
picro-fuchsin, van Gieson's, 79 
picro-nigrosin, 79 
Ranvier's formic acid, 102 
Robertson's modification of Hel- 
ler's, for myelin-sheath, 141 
Roehl's, for calcium deposits, 187 
Romanowsky's, for malarial organ- 
isms, 389, 390, 391, 418 
safranin, 77 

for karyomitotic figures, 99 
Schaffer's, for differentiating 
bone from cartilage, 158 
Sahli's borax methylene-blue, 75 
Schaffer's safranin, for differentiat- 
ing bone from cartilage, 158 
Scharlach R. for fat, 165, 167 

for fresh tissue, 34 
Schmorl's, for differentiating bone 

from cartilage, 159 
Schridde's, for granules in cyto- 
plasm of plasma-cells and lym- 
phocytes, no 
for myelocytes and leucocytes, 
150 
Schiitz's, for glanders bacillus, 318 
silver, 99 



INDEX. 



553 



Stains, silver, Von Kossa's, for cal- 
cium deposits, 188 
Smith's, for bacteria in sputum, 

237 ' 
for capsules in sections, 251 
of bacteria for smears, 244 

special, 107 

Sternberg and Eyenes', for trepo- 
nema pallidum in sections, 402 

Stirling's gentian-violet, 77 

Stroebe's anilin blue, for nerve- 
fibers, 134 

Sudan III, for fat, 165 

thionin, Hoyer's, for mucin, 173 

tubercle bacillus method, bacteria 
stained by, 351 

Unna's, for colloid, 176 
for connective tissue, 113 
for elastic fibers, 117 
for hyalin, 176 
for mast-cells, 109, no 
for plasma-cells, no 
methyl ene-blue, alkaline, 74 
polychrome, 74 
for mucin, 173 
methylene-blue, 173 

Unna-Pappenhein's methyl-green- 
pyronin, no, 249 

van Gieson's diffuse, method, 96 
for connective tissue, 112 
for nervous system, 122 
picro-fuchsin, 79 

VerhoefFs, for elastic tissue, 118 
modified Gram for leptothrix of 
Parinaud's conjunctivitis, 250 

von Kossa's silver, for calcium de- 
posits, 188 

Weigert's alcohol hematoxylin, 70 
for fibrin, 1 70 
for my elm-sheath, 135 

Kulschitzky-Wolter's modifi- 
cation, 139 
Pal's modification, 138 
for neuroglia-fibers, 144 
iron hematoxylin, 89 

Weigert-Gram, for bacteria, 250 

Williams and Lowden, for Negri 
bodies, 395 

Wright's, for blood, 106, 418 
for blood-films, microscopic ap- 
pearances, 420 
for bone-marrow, 149 
for malarial parasites, 389 
for treponema pallidum, 397 
myelin-sheath, for frozen sec- 
tions, 141 

Zettnow's, for flagella, 244 

Ziehl-Neelson's carbol-fuchsin, 75 

Ziehl-Neelson-Gabbet, for tubercle 
bacillus, 352 



Staphylococcus cereus albus, 257 
flavus, 257 
epidermidis albus, 256 
pyogenes albus, 256 
aureus, 253 

diagnosis, 256 
citreus, 256 

gonococcus and, differentiation, 
274 
Starch-granules, iodin and sulphuric 

acid, for 181 
Stearin, 165 
Stender dishes, 30 
Sterilization of culture-media, 206 

autoclave for, 207 
Sternberg and Eyenes' method for 
staining treponema pallidum in 
sections, 402 
Sternberg's sputum-septicemia, 266 
Stirling's gentian-violet, 77 
Stomach, 154 
contents, 469. See also Gastric 

contents. 
hemorrhage from, autopsy guides, 

479 
opening of, 498 
removal of, 497 
Storage of microscope slides, 31 
Streptococcus brevis, 258 
capsulatus, 270 
conglomeratus, 258 
longus, 258 
pyogenes, 257" 

and allied streptococci, 257 

diagnosis, 263 

gonococcus and, differentiation, 

274 _ 

pneumococcus and, differentia- 
tion, 258 
viridans, 260 
Striated muscle-cells, staining, 119 
Stroebe's aniline blue for nerve-fibers, 

i34 
Stropping, 28 

Subcutaneous inoculation of guinea- 
pigs, 229 
Sudan III for fat, 165 
Sudden death, autopsy guides, 479 

opening heart in, 490 
Sugar agar for actinomyces bovis, 367 
Sulphuric acid and iodin for amyloid, 
181 
for cellulose, 181 
for cholesterin crystals, 181 
for corpora amylacea, 181 
for starch-granules, 181 
Sulphurous acid in decalcification, 52 
Suspension, 233 

Suzuki's method for serial celloidin 
sections, 66 



554 



INDEX. 



Swabs, 210 

Syphilis, Noguchi's reaction in, 427. 
See also Noguchi reaction. 
of central nervous system, Lange's 
colloidal gold test of cerebro- 
spinal fluid for, 451 
Staining, 397 

Wassermann reaction in, 427. See 
also Wassermann reaction. 
Syracuse solid watch-glasses, 29 

Table tops, method for blackening, 

529 
Taenia echinococcus, 405 

mediocanellata solium saginata, 
405 

solium, 404 
Tape-worms, 403 
Tea infuser, Hobb's, 30 
Teased preparations, ^ 
Tellyesniczky's mixture as fixative, 

47 
Terminal infection, 262 
I processes, stains for, 129 
Tertian parasite, 385 

cycle of, 387 
Testicles, removal of, 503 

weight of, 503 
Test-tubes, filling, 204 

preparation of, for cultures, 189 

quantity of medium in, 205 
Tetanus bacillus, 336 

toxin of, 339 
Thionin, Hoyer's, for mucin, 173 
Thoma-Zeiss hemocytometer, 410, 

411 
Thoracic duct, position of, 504 

organs, removal of, 492 
Thorax, incision to bare, 483, 485 

opening of, 483, 485 
Thyme, oil of, as clearing reagent, 
104 
and cloves, as clearing reagent, 
105 
Tigroid bodies, stains for, 1 24 
Tincture of iodin as stain, 83 
Tissue, fresh. See Fresh tissue. 

from clinical cases, examination for 
diagnosis, 454 

tubercle bacilli in, 350 
Tissue-elements, those stained, 85 
Titration in adjustment of media re- 
action, 202 
Topfer's dimethyl-amido-azo-benzol 

test for hydrochloric acid, 470 
Toxalbumin of bacillus diphtherias, 

307 
tetanus, 339 
Toxin, bacillus diphtherias, 307 
tetanus, 339 



Transplanting culture, 216 
1'ransudations, examination, 455 
Treponema pallidum, Benian's meth- 
od, 399 
Burri's India-ink method, 401 
Eyenes and Sternberg's stain, 

402 
Fontana's method, 399 
. Ghoreyeb's stain, 400 
Giemsa's stain, 397 
in smear preparations, 397 
Levaditi's stain, 401 
Wright's stain, 397 
Trichinellae, 408 

embryo, 409 
Trichloracetic acid in decalcification, 

5 2 
Tubercle bacillus, 342. See also 

Bacillus tuberculosis. 
Tubercles, glanders, 316 
Tuberculosis, autopsy guides, 478 

bacillus of, 342. See also Bacillus 
tuberculosis. 
Turnbull's blue, 186 
Twine, hemp, 476 
Typhoid fever, bacillus of, 293. See 

also Bacillus typhosus. 

Umbilical arteries, examination, 520 

cord, examination, 520 
Unna-Pappenheim's methyl-green 
pyronin stain for bacteria, 
249 
for plasma : cells, no 
Unna's method for mast-cells, 109, no 
methylene-blue, alkaline, 74 

polychrome, 74, 173 
orcein, for connective tissue, 113 

for elastic fibers, 117 
polychrome methylene-blue for 

mucin, 173 
stain for colloid, 176 

for connective tissue, 113 
for hyalin, 176 
for plasma-cells, no 
Urinary tract, removal of, 501 
Urine, bacillus tuberculosis in, 350 

examination of, 471 
Uterine scrapings, examination, 454 
Uterus, opening of, 503 

Vaccines, bacterial, of Wright, prep- 
aration, 525 

Vagina, incision of, 503 

Valvular competence, water-test for, 
490 

Van Gieson's connective tissue stain, 
112 
diffuse stain, method, 96 
nervous tissue stain, 122 



INDEX. 



555 



Van Gieson's picro-fuchsin, 79 
Vegetable parasites in sputum, 237, 

466 
Vena cava, inferior, opening of, 504 
Ventricles, opening of, 489 
Verhoeff's carbol-fuchsin solution, 75 

elastic tissue stain, 118 

method for serial colloidin sections, 

modified Gram stain for leptothrix 
of Parinaud's conjunctivitis, 250 

Verocay's method of removing pre- 
cipitate from formaldehyde, 42 

Vertebral column, removal of, 505 

Vibrio, cholera, 355. See also Com- 
ma bacillus. 

Virchow's method for examining 
brain, 513 

Von Kossa's silver stain for calcium 
deposits, 188 

Vulcanized fiber for mounting, 26 

Wassermann reaction in syphilis, 427 
amboceptor, 428, 433 

antihuman, 433 

antisheep, 435 

standardization, 437 
antigen, 429, 435 

standardization, 440 
apparatus needed, 432 
centrifuge in, 433 
complement, 427, 433 
corpuscle suspension, 437 
interpretation of results, 441 
patient's serum, 436 
preparation of reagents, 43 2 
saline solution in, 433 
standardization of reagents, 

437 
technique, 441 
water-bath in, 433 
Wright's modification, 445 
Watch-glasses, solid, Syracuse, 29 
Water, running, for washing speci- 
mens, 28 
Water-bath in serum diagnosis of 

syphilis, 433 
Water-test of valvular competence, 

490 
Wedge-shaped incision for opening 

skull, 506 
Weigert's alcohol hematoxylin, 70 
differential stain for fibrin, 170 
iron hematoxylin, 89 
method for serial celloidin sections. 

64 
mixtures as clearing reagents, 105 
stain for elastic fibers, 116 

Hart's modification, 117 
for myelin-sheath, 135 



Weigert's stain for myelin-sheath, 
Kulschitzky-Wolter's modi- 
fication, 139 
Pal's modification, 138 
for neuroglia-fibers, 144 
Weigert-Gram method for bacteria, 
250 
for scarlet fever, 313 
White blood-globules in sputum, 465 

corpuscles. See Leucocytes. 
Whooping-cough, bacillus of, 286 
Widal's method for examination of 
serous fluids, 456, 457 
reaction, 297 
Williams and Lowden's method for 

Negri bodies, 395 
Williams' method for staining flagella, 

241 
Wolbach's modification of Giemsa's 
stain for protozoa and bacteria in 
sections, 393 
Wool-sorters' disease, bacillus of, 327 

diagnosis, 330 
Worms, round-, 406 

tape-, 403 
Wounds inflicted at autopsy, treat- 
ment, 478 
Wright's bacterial vaccines, prepara- 
tion, 525 
embedding method for frozen sec- 
tions, 54 
method for anaerobes, 225 

of counting blood-platelets, 414 
modification of Wassermann reac- 
tion in syphilis, 445 
myelin-sheath stain for frozen sec- 
tions, 141 
stain for blood, 106, 418 

for blood-films, microscopic ap- 
pearances after, 420 
for bone-marrow, 150 
for malarial parasites, 389 
for treponema pallidum, 397 

Xylol and aniline as clearing re- 
agent, 105 

and carbolic acid as clearing re- 
agent, 105 

as clearing reagent, 104 

balsam, 106 

Zenker's fluid as fixative, 45, 247 
Helly's modification, 38, 46 

Zettnow's method for staining fla- 
gella, 244 

Ziehl-Neelson's carbol-fuchsin, 75 

Ziehl-Neelson-Gabbet method for tu- 
bercle-bacillus, 352 

Zinsser's method for anaerobic plate- 
cultures, 224 



